Epithelial immunomodulation by aerosolized Toll-like receptor agonists prevents allergic inflammation in airway mucosa in mice

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 (“Pam2”, TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 (“ODN”, TLR9 ligand), when delivered together by aerosol (“Pam2ODN”), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy

Interleukins-4, -5, and -13: emerging therapeutic targets in allergic disease

For the first time, allergic diseases have emerged as major public health concerns. Highly effective therapies for allergic disease now exist, but are plagued by serious side effects and the fact that a significant minority of patients remains unresponsive. Studies from many laboratories have established that T helper type 2 (TH2) cytokines contribute importantly to diseases such as asthma, and therapeutic strategies that target the key TH2 cytokines are of potential benefit in allergic disease. In this article, we will review the biology of the TH2 cytokines interleukin (IL)-4, IL-5, and IL-13 and their receptors, and will consider several novel strategies to neutralize these molecules in human and experimental asthma. While promising, newer therapies face a gauntlet of developmental challenges, but offer the hope of reducing allergic diseases once again to minor public health concerns

Correction: Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence.

[This corrects the article DOI: 10.1371/journal.ppat.1005678.]

Separate endocytic pathways regulate IL-5 receptor internalization and signaling

Eosinophils are critically dependent on IL-5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, βc, which is shared by IL-5, IL-3, and GM-CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2-dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of βc-sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL-5Rs reside in and are internalized by clathrin- and lipid raft-dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of βc, IL-5Rα, and Cy3-labeled IL-5 with transferrin- (clathrin) and cholera toxin-B- (lipid raft) positive vesicles. Moreover, whereas internalized IL-5Rs were detected in both clathrin- and lipid raft-positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome-degraded IL-5Rs partitioned to the soluble, nonraft fractions (clathrin-containing). Lastly, we show that optimal IL-5-induced signaling requires entry of activated IL-5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL-5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL-5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL-5R endocytosis regulates signaling both positively and negatively

BclA-mediated CFH recruitment inhibited downstream complement activation <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Complement hemolytic assay. Spores were incubated with 20% NHS and centrifuged. The supernatants (1:10 diluted) were used to perform complement hemolytic assays using opsonized sheep erythrocytes (EA-SRBC). Data shown was from at least three independent experiments. (<b>B</b>) Determination of C5a levels in human serum incubated with the different spores. GVB⁰ buffer containing 20% NHS was pre-treated with buffer only (no antibody), OX24, or control IgG1, followed by incubation with 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores. C5a levels in the supernatants were measured using the Human Complement Component C5a DuoSet. Data shown was combined from two independent experiments, each with duplicate wells. (<b>C</b>) Determination of C5a levels in mouse BAL fluid. C57BL/6 were i.n. inoculated with 7702 (n = 8), Δ<i>bclA</i> (n = 8), Δ<i>bclA</i>/BclA (n = 8) spores or PBS (n = 6). BAL fluids were collected 6 hours later and C5a level in the supernatant determined using the Mouse Complement Component C5a DuoSet. Data shown were combined from two independent experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01. ****, <i>p</i> < 0.0001, <i>t</i> test.</p

BclA-mediated CFH binding promoted degradation of C3b on the spore surface and downregulated further C3 activation.

<p>(<b>A</b>) and (<b>B</b>) Spores were incubated with purified human C3b, CFH and CFI. C3 fragments deposited on the spore surface were detected using anti-C3 polyclonal antibodies (<b>A</b>). The image shown is representative of at least three independent experiments. The ratio of iC3b/C3b was determined by quantifying the density of the corresponding bands in western blots using Image J (<b>B</b>). The β chain represents C3b + iC3b, and the α” chain represents iC3b. Results were combined from three independent experiments. (<b>C</b>) and (<b>D</b>) Rate of iC3b deposition on spores. Spores were incubated with 10% NHS for the indicated time and subjected to flow cytometry analysis using iC3b-specific antibody. The results shown are mean fluorescence intensity normalized to that at 10 minutes, respectively, and combined from at least three independent experiments (<b>C</b>). The rate of iC3b deposition (ΔiC3b/Δt) was calculated by linear regression analysis of the normalized data (GraphPad Prism 6) (<b>D</b>). (<b>E</b>) Determination of C3a concentration. GVB⁰ buffer containing 20% NHS (no antibody), 20% NHS pre-treated with the CFH functional blocking antibody OX24 (240 nM final conc.) or mouse IgG1 control (240 nM) was incubated with no spore, 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores at 37°C for 30 min and centrifuged to remove the spores. C3a concentrations in the supernatants were determined using the Human C3a ELISA kit (BD OptEIA^™) and normalized to the respective no spore control. Data was combined from four experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; <i>t</i> test.</p

BclA inhibited antibody responses against spores.

<p>(<b>A</b>) C57BL/6 mice were i.n. inoculated with ~1×10⁸ spores of 7702, Δ<i>bclA</i> or vehicle control once and blood collected at 2 weeks post inoculation (2wk), or inoculated again with the same spores and dose at 2 weeks and blood collected at 4 weeks after the initial inoculation (4wk). Anti-spore antibodies in the serum were detected using ELISA. Data shown were combined from at least three independent experiments. The mouse number for the various groups is as follows: control, n = 8; 2wk experiment, n = 30 and 29 for 7702 and Δ<i>bclA</i>, respectively; 4wk experiment, n = 30 and 28 for for 7702 and Δ<i>bclA</i>, respectively. (<b>B</b>) C3^-/- mice were i.n. inoculated with vehicle control, or ~5×10⁵ spores of 7702 or Δ<i>bclA</i> and blood collected at 2 weeks post inoculation. Anti-spore antibodies in the serum were detected using ELISA. Data shown were combined from at least three independent experiments, with n = 10, 24 and 21 for control, 7702 and Δ<i>bclA</i>, respectively. *, <i>p</i> < 0.05; **, <i>p <</i> 0.01; ****, <i>p</i> < 0.0001; <i>t</i> test.</p