Modulatory Role for Retinoid-related Orphan Receptor α in Allergen-induced Lung Inflammation

Rationale: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma

Pulmonary Immune Responses to Propionibacterium acnes in C57BL/6 and BALB/c Mice

Propionibacterium acnes (PA) is a gram-positive anaerobic bacterium implicated as a putative etiologic agent of sarcoidosis. To characterize the pulmonary immune response to PA, C57BL/6 and BALB/c mice were intraperitoneally sensitized and intratracheally challenged with heat-killed bacteria. C57BL/6 mice challenged with PA developed a cellular immune response characterized by elevations in Th1 cytokines/chemokines, increased numbers of lymphocytes and macrophages in lung lavage fluid, and peribronchovascular granulomatous inflammation composed of T- and B-lymphocytes and epithelioid histiocytes. T-lymphocytes in the lung lavage fluid showed a marked CD4+ cell predominance. In contrast, C57BL/6 mice challenged with Staphylococcus epidermidis (SE), another gram-positive commensal of human skin, and BALB/c mice challenged with PA, showed only a modest induction of Th1 cytokines, less pulmonary inflammation, and no granulomatous changes in the lung. Enhancement of Toll-like receptor expression was seen in PA-exposed C57BL/6 mice within 24 h after exposure, suggesting that induction of innate immunity by PA contributes to the robust, polarized Th1 immune response elicited by this bacterium. These findings suggest that PA-induced pulmonary inflammation may be a useful model for testing the contributions of both bacterial and host factors in the development, maintenance, and resolution of granulomatous inflammation in the lung

Modulatory Role for Retinoid-related Orphan Receptor α in Allergen-induced Lung Inflammation

Rationale: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma. Objective: In this study, we assess the potential regulatory function of retinoid-related orphan receptor α (RORα) in the adaptive immune response using ovalbumin (OVA)-induced airway inflammation as a model. Methods: Allergen-induced inflammation was compared between wild-type (WT) and staggerer (RORα(sg/sg)) mice, a natural mutant strain that is deficient in RORα expression. Measurements and Main Results: Despite robust increases in OVA-specific IgE, RORα(sg/sg) mice developed significantly less pulmonary inflammation, mucous cell hyperplasia, and eosinophilia compared with similarly treated WT animals. Induction of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, was also significantly less in RORα(sg/sg) mice. Microarray analysis using lung RNA showed increased expression of many genes, previously implicated in inflammation, in OVA-treated WT mice. These include mucin Muc5b, the chloride channel calcium-activated 3 (Clca3), macrophage inflammatory protein (MIP) 1α and 1β, eotaxin-2, serum amyloid A3 (Saa3), and insulin-like growth factor 1 (Igf1). These genes were induced to a greater extent in OVA-treated WT mice relative to RORα(sg/sg) mice. Conclusions: Our study demonstrates that mice deficient in RORα exhibit an attenuated allergic inflammatory response, indicating that RORα plays a critical role in the development of Th2-driven allergic lung inflammation in mice, and suggests that this nuclear receptor should be further evaluated as a potential asthma target

Structural and Biophysical Characterization of <i>Bacillus thuringiensis</i> Insecticidal Proteins Cry34Ab1 and Cry35Ab1

<div><p><i>Bacillus thuringiensis</i> strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from <i>B. thuringiensis</i> strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) <i>Diabrotica virgifera virgifera</i> Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.</p></div

<b>Table 2.</b> Cry35Ab1 (4JP0) data processing, model and refinement statistics.

a<p> R_merge  =  100Σ(h)Σ(i)|I(i)-<i>|/Σ(h)Σ(i)I(i) where I(i) is the <b>i</b>th intensity measurement of reflection h, and <i> is the average intensity from multiple observations.</i></i></p><i><i>b<p> R_cryst  =  Σ||<b>F</b>_obs|-|<b>F</b>_calc||/Σ|<b>F</b>_obs|. Where <b>F</b>_obs and <b>F</b>_calc are the structure factor amplitudes from the data and the model, respectively. R_free is R_cryst with 10% of the structure factors.</p>c<p> Number of residues in favored/additionally favored outlier region. Calculated using PROCHECK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Laskowski1" target="_blank">[14]</a>.</p><p><b>Table 2.</b> Cry35Ab1 (4JP0) data processing, model and refinement statistics.</p></i></i

Crystal structures of Cry34Ab1 and Cry35Ab1.

<p>(A) The structure of Cry34Ab1 is a β-sandwich of 10 strands. (B) Cry35Ab1 contains two domains. The N-terminal trefoil domain contains α-helices and three β-sheets. The C-terminal domain is terminated with a three helix fold which is not required for activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Gao1" target="_blank">[17]</a>. This figure, and all subsequent structure representations, were made with PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-DeLano1" target="_blank">[66]</a>.</p

Comparison of proteins structurally related to Cry35Ab1.

<p>Cry35Ab1 is structurally related to a wide variety pore-forming proteins as assessed by combinatorial extension. All structures contain a conserved beta-sheet core and varying loop regions.</p

<b>Table 1.</b> Cry34Ab1 (4JOX) data processing, model and refinement statistics.

a<p> R_merge  =  100Σ(h)Σ(i)|I(i)-<i>|/Σ(h)Σ(i)I(i) where I(i) is the <b>i</b>th intensity measurement of reflection h, and <i> is the average intensity from multiple observations.</i></i></p><i><i>b<p> R_cryst  =  Σ||<b>F</b>_obs|-|<b>F</b>_calc||/Σ|<b>F</b>_obs|. Where <b>F</b>_obs and <b>F</b>_calc are the structure factor amplitudes from the data and the model, respectively. R_free is R_cryst with 10% of the structure factors.</p>c<p> Number of residues in favored/additionally favored outlier region. Calculated using PROCHECK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Laskowski1" target="_blank">[14]</a>.</p><p><b>Table 1.</b> Cry34Ab1 (4JOX) data processing, model and refinement statistics.</p></i></i

<b>Table 5.</b> Combinatorial extension analysis of PDB submitted structures against Cry35Ab1 coordinates.

<p>Results of the top 10 unique hits ranked according to Z-score.</p><p><b>Table 5.</b> Combinatorial extension analysis of PDB submitted structures against Cry35Ab1 coordinates.</p