Biological effects of inhaled crude oil. VI. Immunotoxicity.

Crude oil is an unrefined petroleum product that is a mixture of hydrocarbons and other organic material. Studies on the individual components of crude oil and crude oil exposure itself suggest it has immunomodulatory potential. As investigations of the immunotoxicity of crude oil focus mainly on ingestion and dermal exposure, the effects of whole-body inhalation of 300 ppm crude oil vapor [COV; acute inhalation exposure: (6 h × 1 d); or a 28 d sub-chronic exposure (6 h/d × 4 d/wk. × 4 wks)] was investigated 1, 28, and 90 d post-exposure in Sprague-Dawley rats. Acute exposure increased bronchoalveolar lavage (BAL) fluid cellularity, CD4+ and CD8+ cells, and absolute and percent CDllb+ cells only at 1 d post-exposure; additionally, NK cell activity was suppressed. Sub-chronic exposure resulted in a decreased frequency of CD4+ T-cells at 1 d post-exposure and an increased number and frequency of B-cells at 28 d post-exposure in the lung-associated lymph nodes. A significant increase in the number and frequency of B-cells was observed in the spleen at 1 d post-exposure; however, NK cell activity was suppressed at this time point. No effect on cellularity was identified in the BALF. No change in the IgM response to sheep red blood cells was observed. The findings indicate that crude oil inhalation exposure resulted in alterations in cellularity of phenotypic subsets that may impair immune function in rats.

Potential classification of chemical immunologic response based on gene expression profiles.

Occupational immune diseases are a serious public health burden and are often a result of exposure to low molecular weight (LMW) chemicals. The complete immunological mechanisms driving these responses are not fully understood which has made the classification of chemical allergens difficult. Antimicrobials are a large group of immunologically-diverse LMW agents. In these studies, mice were dermally exposed to representative antimicrobial chemicals (sensitizers: didecyldimethylammonium chloride (DDAC), ortho-phthalaldehyde (OPA), irritants: benzal-konium chloride (BAC), and adjuvant: triclosan (TCS)) and the mRNA expression of cytokines and cellular mediators was evaluated using real-time qPCR in various tissues over a 7-days period. All antimicrobials caused increases in the mRNA expression of the danger signals Tslp (skin), and S100a8 (skin, blood, lung). Expression of the TH2 cytokine Il4 peaked at different timepoints for the chemicals based on exposure duration. Unique expression profiles were identified for OPA (Il10 in lymph node, Il4 and Il13 in lung) and TCS (Tlr4 in skin). Additionally, all chemicals except OPA induced decreased expression of the cellular adhesion molecule Ecad. Overall, the results from these studies suggest that unique gene expression profiles are implicated following dermal exposure to various antimicrobial agents, warranting the need for additional studies. In order to advance the development of preventative and therapeutic strategies to combat immunological disease, underlying mechanisms of antimicrobial-induced immunomodulation must be fully understood. This understanding will aid in the development of more effective methods to screen for chemical toxicity, and may potentially lead to more effective treatment strategies for those suffering from immune diseases.

Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction.

5-Chloro-2-(2,4-dichlorophenoxy)phenol (triclosan) is an antimicrobial chemical widely used in consumer household and clinical healthcare products. Human and animal studies have associated triclosan exposure with allergic disease. Mechanistic studies have identified triclosan as a mitochondrial uncoupler; recent studies suggest that mitochondria play an important role in immune cell function and are involved in activation of the NLRP3 inflammasome. In this study, early immunological effects were evaluated via NLRP3 activation following dermal triclosan application in a BALB/c murine model. These investigations revealed rapid caspase-1 activation and mature IL-1ß secretion in the skin and draining lymph nodes (dLNs) after 1.5% and 3% triclosan exposure. Correspondingly, pro-Il-1b and S100a8 gene expression increased along with extracellular ATP in the skin. Peak gene expression of chemokines associated with caspase-1 activation occurred after 2 days of exposure in both skin tissue and dLNs. Phenotypic analysis showed an increase in neutrophils and macrophages in the dLN and myeloid and inflammatory monocytes in the skin tissue. Triclosan also caused mitochondrial dysfunction shown through effects on mitochondrial reactive oxygen species, mass, mitochondrial membrane potential, and mitochondrial morphology. These results indicate that following triclosan exposure, activation of the NLRP3 inflammasome occurs in both the skin tissue and dLNs, providing a possible mechanism for triclosan's effects on allergic disease and further support a connection between mitochondrial involvements in immunological responses.

Long-term Immunotoxic Effects of Oral Prenatal and Neonatal Atrazine Exposure.

Atrazine and its metabolites are present at high concentrations in many water supplies in agro-intensive areas. Because residents in these areas drink water from sources fed from these contaminated supplies, we investigated the long-term immunotoxicity of combined prenatal and neonatal (perinatal) exposure to atrazine via drinking water, on the immune system in mice. At 6 months of age, upon immunization with heat-killed Streptococcus pneumoniae, the serum IgG antibody response against the T independent antigen phosphorylcholine was significantly higher in male, but not female, atrazine-exposed mice as compared with that in untreated controls. No alterations were present in all offspring in the serum antibody response against the T-dependent antigen pneumococcal surface protein A (PspA). ELISpot analysis showed only a small, insignificant reduction in PspA-specific IgG producing splenocytes in atrazine-treated male offspring. Interestingly, upon ex vivo stimulation with anti-CD3 and anti-CD28 antibodies, significant decreases in interleukin (IL)-2, tumor necrosis factor-α, interferon-γ, and IL-17A and a decreasing trend in IL-10 were observed in splenocytes from atrazine-exposed male, but not female mice. Analysis of thymic and splenic cell populations showed no effects of atrazine exposure in either sex. This is the first time that long-term changes in the immune response were observed after a perinatal exposure to atrazine and it demonstrates that these early life exposures can result in permanent changes to the immune system as well as a male bias in these effects.

Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway.

The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfα, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (TH2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.

Immune stimulation following dermal exposure to unsintered indium tin oxide.

In recent years, several types of pulmonary pathology, including alveolar proteinosis, fibrosis, and emphysema, have been reported in workers in the indium industry. To date, there remains no clear understanding of the underlying mechanism(s). Pulmonary toxicity studies in rats and mice have demonstrated the development of mediastinal lymph node hyperplasia and granulomas of mediastinal lymph nodes and bronchus-associated lymphoid tissues following exposure to indium tin oxide. Given the association between exposure to other metals and the development of immune-mediated diseases, these studies were undertaken to begin to investigate the immuno-modulatory potential of unsintered indium tin oxide (uITO) in a mouse model. Using modifications of the local lymph node assay, BALB/c mice (five animals/group) were exposed topically via intact or breached skin or injected intradermally at the base of the ear pinnae with either vehicle or increasing concentrations 2.5-10% uITO (90:10 indium oxide/tin oxide, particle size <50 nm). Dose-responsive increases in lymphocyte proliferation were observed with a calculated EC3 of 4.7% for the intact skin study. Phenotypic analysis of draining lymph node cells following intradermal injection with 5% uITO yielded a profile consistent with a T-cell-mediated response. These studies demonstrate the potential for uITO to induce sensitization and using lymphocyte proliferation as a biomarker of exposure, and demonstrate the potential for uITO to penetrate both intact and breached skin.

Toxicological analysis of limonene reaction products using an in vitro exposure system.

Epidemiological investigations suggest a link between exposure to indoor air chemicals and adverse health effects. Consumer products contain reactive chemicals which can form secondary pollutants which may contribute to these effects. The reaction of limonene and ozone is a well characterized example of this type of indoor air chemistry. The studies described here characterize an in vitro model using an epithelial cell line (A549) or differentiated epithelial tissue (MucilAir™). The model is used to investigate adverse effects following exposure to combinations of limonene and ozone. In A549 cells, exposure to both the parent compounds and reaction products resulted in alterations in inflammatory cytokine production. A one hour exposure to limonene+ozone resulted in decreased proliferation when compared to cells exposed to limonene alone. Repeated dose exposures of limonene or limonene+ozone were conducted on MucilAir™ tissue. No change in proliferation was observed but increases in cytokine production were observed for both the parent compounds and reaction products. Factors such as exposure duration, chemical concentration, and sampling time point were identified to influence result outcome. These findings suggest that exposure to reaction products may produce more severe effects compared to the parent compound.

Potential immunotoxicological health effects following exposure to COREXIT 9500A during cleanup of the Deepwater Horizon oil spill.

Workers involved in the Deepwater Horizon oil spill cleanup efforts reported acute pulmonary and dermatological adverse health effects. These studies were undertaken to assess the immunotoxicity of COREXIT 9500A, the primary dispersant used in cleanup efforts, as a potential causative agent. COREXIT 9500A and one of its active ingredients, dioctyl sodium sulfosuccinate (DSS), were evaluated using murine models for hypersensitivity and immune suppression, including the local lymph node assay (LLNA), phenotypic analysis of draining lymph node cells (DLN), mouse ear swelling test (MEST), total serum immunoglobulin E (IgE), and the plaque-forming cell (PFC) assay. Dermal exposure to COREXIT 9500A and DSS induced dose-responsive increases in dermal irritation and lymphocyte proliferation. The EC3 values for COREXIT 9500A and DSS were 0.4% and 3.9%, respectively, resulting in a classification of COREXIT 9500A as a potent sensitizer and DSS as a moderate sensitizer. A T-cell-mediated mechanism underlying the LLNA was supported by positive responses in the MEST assay for COREXIT and DSS, indicated by a significant increase in ear swelling 48 h post challenge. There were no marked alterations in total serum IgE or B220+/IgE+ lymph-node cell populations following exposure to COREXIT 9500A. Significant elevations in interferon (IFN)-γ but not interleukin (IL)-4 protein were also observed in stimulated lymph node cells. The absence of increases in IgE and IL-4 in the presence of enhanced lymphocyte proliferation, positive MEST responses, and elevations in IFN-γ suggest a T-cell-mediated mechanism. COREXIT 9500A did not induce immunosuppression in the murine model.

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence.

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin alpha(2)beta(1). Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin alpha2beta(1) and affects the biological outcome of host-pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the alpha2beta(1) integrin, because (i) both adherence and internalization of the scl1-inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-alpha2 integrin-subunit antibody and type I collagen, (iii) recombinant alpha2-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the alpha2beta(1) integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.

Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers.

Collagen triple helix, composed of the repeating Gly-Xaa-Yaa (GXY) sequence, is a structural element found in all multicellular animals and also in some prokaryotes. Long GXY polymers are highly regarded components used in food, cosmetic, biomedical, and pharmaceutical industries. In this study, we explore a new concept for the production of recombinant GXY polymers which are based on the sequence of "prokaryotic collagens", the streptococcal collagen-like proteins Scl1 and Scl2. Analysis of 50 Scl variants identified the amino acid distribution and GXY-repeat usage that are involved in the stabilization of the triple helix in Scls. Using circular dichroism spectroscopy and electron microscopy, we show that significantly different recombinant rScl polypeptides form stable, unhydroxylated homotrimeric triple helices that can be produced both intra- and extracellularly in the Escherichia coli. These rScl constructs containing 20 to 129 GXY repeats had mid-point melting temperatures between 32 and 39 degrees C. Altogether, Scl-derived collagens, which are different from the mammalian collagens, can form stable triple helices under physiological conditions and can be used for the production of recombinant GXY polymers with a wide variety of potential applications.