Development of a Bioluminescent Sensor of Isothiocyanates through Genetic Modification of Pseudomonas fluorescens and Validation on Crude Plant Materials

Isothiocyanates (ITCs) are produced by roots in a variety of agricultural plants, such as those within the Brassicaceae family. ITCs act as fumigants, and are used to control microbial plant pathogens by adding brassica-origin seed meal to soil prior to replanting. However, there is suggestion that the presence of residual ITCs in soil may inhibit seed germination in recently planted crops. These chemical compounds are difficult to detect, where a biological sensor may be a strategic indicator for when the soil is safe to replant in. Some microbes, including Pseudomonas fluorescens, exhibit resistance to ITCs mediated by an induced biochemical pathway. P. fluorescens is a root-colonizing non-pathogenic microbe, making it an ideal and safe biological sensor for agricultural use. To accomplish this, we linked an ITC-responsive promoter (saxA promoter) to the ilux operon, an enhanced version of the lux operon with an additional FMN reductase. The resulting biosensor was responsive in a dose-dependent manner to the aliphatic ITC, sulforaphane, in the concentration range of 1-100 μM. Further tests were conducted using extracted fluid from plant material of broccoli and daikon for the experimental group, while utilizing clover, alfalfa, and mung bean as a negative control group. The biosensor was responsive to the Brassica-related plants, and did not luminesce in response to the negative control extracts. The biosensor was also responsive to the seed meal of Brassica juncea and Sinapis alba, which are common seed meals used in biofumigation. Future experiments will determine the biosensor’s responsiveness to other ITCs and their precursors, glucosinolates, and detection in seed meal amended soils

Interleukin-33 contributes to both M1 and M2 chemokine marker expression in human macrophages

Abstract Background Interleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined. Results Human macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone. Conclusions Together, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.http://deepblue.lib.umich.edu/bitstream/2027.42/78250/1/1471-2172-11-52.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78250/2/1471-2172-11-52.pdfPeer Reviewe

A systemic granulomatous response to Schistosoma mansoni eggs alters responsiveness of bone marrow‐derived macrophages to Toll‐like receptor agonists

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141008/1/jlb0314.pd

Macrophage/fibroblast coculture induces macrophage inflammatory protein‐1a production mediated by intercellular adhesion molecule‐1 and oxygen radicals

This study examined the cell‐to‐cell interaction between fibroblasts and macrophages as a possible contributor to the chronic inflammatory state. In a coculture system, consisting of macrophages layered over confluent fibroblasts, there was a significant increase in macrophage inflammatory protein 1α (MIP‐1α) compared with control cultures. ICAM‐1 adhesion was identified as an important stimulus of MIP‐1α production by using ICAM‐1‐specific monoclonal antibodies. Furthermore, fibroblasts from ICAM‐1 knockout mice induced significantly less MIP‐1α production from peritoneal macrophages when compared to control fibroblasts. In addition, it appeared that oxygen radicals functioned as activating molecules during cellular interaction and production of MIP‐1α, as the addition of the antioxidant N‐acetylcysteine (NAC) prevented MIP‐1α secretion. Thus, the ICAM‐1 and oxygen radical‐mediated induction of MIP‐1α associated with a macrophage/fibroblast coculture system provides one possible mechanism by which immune/inflammatory cell interactions may augment chemokine production and exacerbate chronic inflammatory diseases. J. Leukoc. Biol. 64: 636–641; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141120/1/jlb0636.pd

Cell‐to‐cell and cell‐to‐matrix interactions mediate chemokine expression: an important component of the inflammatory lesion

Although many studies have characterized soluble factors that stimulate or inhibit chemokine secretion, in this review we focus on the event of cellular adhesion as a novel mechanism for stimulating chemokine expression. Recent work has demonstrated chemokine expression following cell‐to‐cell and cell‐to‐matrix adhesion. The specificity of this finding was demonstrated utilizing various techniques that illustrate that adhesion, and not a soluble stimulus, is in some cases responsible for initiating or augmenting chemokine expression. For example, co‐cultures of peripheral blood monocytes and endothelial cells secreted elevated levels of IL‐8 and MCP‐1 compared with either cell type alone. When co‐cultured in transwells, this effect was significantly attenuated. In other experiments, neutralizing monoclonal antibodies to various adhesion molecules inhibited chemokine expression. The effects of adhesion were not limited to leukocytes. Both immune and non‐immune cell types were evaluated as potential sources of adhesion‐mediated chemokine expression. Not suprisingly, expression of some chemokines was associated with adhesion, whereas others were not, supporting the notion that adhesion differentially signals chemokine secretion during the inflammatory response. We hypothesize that as a recruited leukocyte encounters different adhesion substrates such as endothelial cells, basement membrane, extracellular matrix, and fibroblasts, the expression of chemokines from both the leukocyte and the substrate may be initiated, inhibited, or augmented. Careful characterization of the contribution of adhesion to regulation of chemokine expression will provide insight into the pathogenesis of many human diseases where chemokines have a central role. J. Leukoc. Biol.62: 612–619; 1997.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142209/1/jlb0612.pd

IL-18 modulates chronic fungal asthma in a murine model; putative involvement of Toll-like receptor-2

Fungus-induced asthmatic disease is characterized by persistent airway hyperreactivity and remodeling.¶ Objective and design: To determine the role of IL-18 in the allergic airway response to Aspergillus fumigatus conidia in a murine model of A. fumigatus -induced asthma.¶ Methods: A. fumigatus -sensitized mice were depleted of IL-18 using a polyclonal anti-IL-18 antibody for 3 days after a conidia challenge.¶ Results: Airway hyperresponsiveness and eosinophil numbers were significantly elevated 3-30 days after conidia challenge compared to the normal serum-treated group. Histological evidence showed retention of A. fumigatus conidia, airway remodeling, subepithelial fibrosis, and increased collagen deposition in the lungs of IL-18-depleted mice at day 30 after the conidia challenge. Prolonged retention of conidia in IL-18 depleted A. fumigatus -sensitized mice was associated with decreased Toll-like receptor-2 (TLR-2) expression compared with the control group.¶ Conclusions: IL-18 modulates the innate immune response against A. fumigatus conidia and prevents the development of severe fungus-induced asthmatic disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41823/1/11-50-11-552_10500552.pd

Acute inhibition of nitric oxide exacerbates airway hyperresponsiveness, eosinophilia and C-C chemokine generation in a murine model of fungal asthma

Objective and Design: This study examined he role of nitric oxide in changes in airway physiology and inflammation in a murine model of fungal allergy induced by Aspergillus fumigatus (A. fumigatus) by treatment of A. fumigatus-sensitized mice with NG-nitro-L-arginine methyl ester (L-NAME) or D-NAME (8 mg/kg; i.p.).¶ Materials and Methods: Female CBA/J mice received A. fumigatus antigen dissolved in incomplete Freund's adjuvant (10 mg/100 ml i.p. and s.c.) followed 2 weeks later by A. fumigatus antigens (20 mg; i.n.) and a subsequent i.t. challenge 4 days later. Airway physiology and inflammation were examined (24 to 72 h) following i.t. challenge.¶ Results: L-NAME-treated mice had lower lung nitrite levels 24 h after A. fumigatus challenge, but higher airway hyperresponsiveness and inflammation compared to D-NAME controls. Airway inflammation in the L-NAME treatment group (72 h) was characterized by a greater bronchoalveolar lavage (BAL), peribronchial eosinophilia and augmented levels of CC chemokines compared to controls.¶ Conclusions: These findings suggest that nitric oxide is an important modulator of airway hyperresponsiveness, inflammation and C-C chemokine generation during allergic airway responses to A. fumigatus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41821/1/11-49-6-297_00490297.pd

Mast cells produce ENA‐78, which can function as a potent neutrophil chemoattractant during allergic airway inflammation

The inflammatory response during allergic airway inflammation involves the recruitment of multiple leukocyte populations, including neutrophils, monocytes, lymphocytes, and eosinophils. All of these populations likely contribute to the pathology observed during repeated episodes of allergic airway inflammation. We have examined the role of a human neutrophil‐specific chemokine (C‐x‐C), ENA‐78, in a model of allergic airway responses and identified murine mast cells as a cellular source of an ENA‐78‐like molecule. Within this allergic airway model, neutrophil infiltration into the airway occurs within 4–8 h post‐allergen challenge, persists within the airway until 24 h, and resolves by 48 h post‐challenge. Neutrophil influx precedes the eosinophil infiltration, which peaks in the airway at 48 h post‐allergen challenge. In this study the production of ENA‐78 from challenged lungs demonstrated a significant increase in the allergen‐,but not vehicle‐, challenged lungs. In vivo neutralization of ENA‐78 by passive immunization demonstrated a significant decrease in peak neutrophil infiltration at 8 h, with no effect on the eosinophil infiltration at 48 h post‐challenge. Because ENA‐78 has been shown to be chemotactic for neutrophils and given the involvement of mast cell degranulation in allergic responses, we examined mast cells for the presence of ENA‐78. Cultured mast cells spontaneously released ENA‐78, but on activation with IgE + antigen, NG‐L‐arginine methyl ester or compound 48/80 produced significantly increased levels of ENA‐78. Supernatants from sonicated MC‐9 mast cells induced an overwhelming influx of neutrophils into the BAL by 4 h post‐intratracheal injection into mice, suggesting that the mast cell is a significant source of neutrophil chemotactic factors. Mast cell supernatant‐mediated neutrophil infiltration was substantially decreased by preincubation of the supernatant with antibodies specific for ENA‐78. These data indicate a major neutrophil chemotactic protein produced by mast cells during allergic responses may be mast cell‐derived ENA‐78. J. Leukoc. Biol. 63: 746–751; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141710/1/jlb0746.pd