Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation.

Heat shock proteins (Hsps) are highly conserved molecular chaperones that are ubiquitously expressed in all species to aid the solubilization of misfolded proteins, protein degradation, and transport. Elevated levels of Hsp70 have been found in the sputum, serum, and bronchoalveolar lavage (BAL) fluid of asthma patients and are known to correlate with disease severity. However, the function of Hsp70 in allergic airway inflammation has remained largely unknown. This study aimed to determine the role of Hsp70 in airway inflammation and remodeling using a mouse model of allergic airway inflammation. WT and Hsp70 double-knockout (Hsp70.1/.3-/-) mice were sensitized and challenged intratracheally with Schistosoma mansoni soluble egg antigens (SEAs) to induce robust Th2 responses and airway inflammation in the lungs. The lack of Hsp70 resulted in a significant reduction in airway inflammation, goblet cell hyperplasia, and Th2 cytokine production, including IL-4, IL-5, and IL-13. An analysis of the BAL fluid suggested that Hsp70 is critically required for eosinophilic infiltration, collagen accumulation, and Th2 cytokine production in allergic airways. Furthermore, our bone marrow (BM) transfer studies show that SEA-induced airway inflammation, goblet cell hyperplasia, and Th2 cytokine production were attenuated in WT mice that were reconstituted with Hsp70-deficient BM, but these effects were not attenuated in Hsp70-deficient mice that were reconstituted with WT BM. Together, these studies identify a pathogenic role for Hsp70 in hematopoietic cells during allergic airway inflammation; this illustrates the potential utility of targeting Hsp70 to alleviate allergen-induced Th2 cytokines, goblet cell hyperplasia, and airway inflammation.

Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni.

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

The NIH-NIAID Schistosomiasis Resource Center at the Biomedical Research Institute: Molecular Redux.

Schistosomiasis remains a health burden in many parts of the world. The complex life cycle of Schistosoma parasites and the economic and societal conditions present in endemic areas make the prospect of eradication unlikely in the foreseeable future. Continued and vigorous research efforts must therefore be directed at this disease, particularly since only a single World Health Organization (WHO)-approved drug is available for treatment. The National Institutes of Health (NIH)-National Institute of Allergy and Infectious Diseases (NIAID) Schistosomiasis Resource Center (SRC) at the Biomedical Research Institute provides investigators with the critical raw materials needed to carry out this important research. The SRC makes available, free of charge (including international shipping costs), not only infected host organisms but also a wide array of molecular reagents derived from all life stages of each of the three main human schistosome parasites. As the field of schistosomiasis research rapidly advances, it is likely to become increasingly reliant on omics, transgenics, epigenetics, and microbiome-related research approaches. The SRC has and will continue to monitor and contribute to advances in the field in order to support these research efforts with an expanding array of molecular reagents. In addition to providing investigators with source materials, the SRC has expanded its educational mission by offering a molecular techniques training course and has recently organized an international schistosomiasis-focused meeting. This review provides an overview of the materials and services that are available at the SRC for schistosomiasis researchers, with a focus on updates that have occurred since the original overview in 2008.

Enhanced protection from fibrosis and inflammation in the combined absence of IL-13 and IFN-γ.

Persistent or dysregulated IL-13 responses are key drivers of fibrosis in multiple organ systems, and this identifies this cytokine as an important therapeutic target. Nevertheless, the mechanisms by which IL-13 blockade leads to the amelioration of fibrosis remain unclear. Because IFN-γ exhibits potent anti-fibrotic activity, and IL-4Rα signalling antagonizes IFN-γ effector function, compensatory increases in IFN-γ activity following IL-13/IL-4Rα blockade might contribute to the reduction in fibrosis. To investigate the role of IFN-γ, we developed novel IL-13(-/-) /IFN-γ(-/-) double cytokine-deficient mice and examined disease progression in models of type 2-driven fibrosis. As predicted, we showed that fibrosis in the lung and liver are both highly dependent on IL-13. We also observed increased IFN-γ production and inflammatory activity in the tissues of IL-13-deficient mice. Surprisingly, however, an even greater reduction in fibrosis was observed in IL-13/IFN-γ double deficient mice, most notably in the livers of mice chronically infected with Schistosoma mansoni. The increased protection was associated with marked decreases in Tgfb1, Mmp12, and Timp1 mRNA expression in the tissues; reduced inflammation; and decreased expression of important pro-inflammatory mediators such as TNF-α. Experiments conducted with neutralizing monoclonal antibodies to IL-13 and IFN-γ validated the findings with the genetically deficient mice. Together, these studies demonstrate that the reduction in fibrosis observed when IL-13 signalling is suppressed is not dependent on increased IFN-γ activity. Instead, by reducing compensatory increases in type 1-associated inflammation, therapeutic strategies that block IFN-γ and IL-13 activity simultaneously can confer greater protection from progressive fibrosis than IL-13 blockade alone. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Induction and activation of latent transforming growth factor-ß1 are carried out by two distinct domains of pregnancy-specific glycoprotein 1 (PSG1).

Pregnancy-specific glycoproteins (PSGs) are a family of Ig-like proteins secreted by specialized placental cells. The PSG1 structure is composed of a single Ig variable region-like N-terminal domain and three Ig constant region-like domains termed A1, A2, and B2. Members of the human and murine PSG family have been shown to induce anti-inflammatory cytokines from monocytes and macrophages and to stimulate angiogenesis. We recently showed that recombinant forms of PSG1 (PSG1-Fc and PSG1-His) and PSG1 purified from the serum of pregnant women are associated with the immunoregulatory cytokine TGF-ß1 and activated latent TGF-ß1. Here, we sought to examine the requirement of specific PSG1 domains in the activation of latent TGF-ß1. Plasmon surface resonance studies showed that PSG1 directly bound to the small latent complex and to the latency-associated peptide of TGF-ß1 and that this binding was mediated through the B2 domain. Furthermore, the B2 domain alone was sufficient for activating the small latent complex. In separate experiments, we found that the PSG1-mediated induction of TGF-ß1 secretion in macrophages was dependent on the N-terminal domain. Mutagenesis analysis revealed that four amino acids (LYHY) of the CC' loop of the N-terminal domain were required for induction of latent TGF-ß1 secretion. Together, our results show that two distinct domains of PSG1 are involved in the regulation of TGF-ß1 and provide a mechanistic framework for how PSGs modulate the immunoregulatory environment at the maternal-fetal interface for successful pregnancy outcome.

RGS16 attenuates pulmonary Th2/Th17 inflammatory responses.

The regulators of G protein signaling (RGS) protein superfamily negatively controls G protein-coupled receptor signal transduction pathways. RGS16 is enriched in activated/effector T lymphocytes. In this paper, we show that RGS16 constrains pulmonary inflammation by regulating chemokine-induced T cell trafficking in response to challenge with Schistosoma mansoni. Naive Rgs16(-/-) mice were "primed" for inflammation by accumulation of CCR10(+) T cells in the lung. Upon pathogen exposure, these mice developed more robust granulomatous lung fibrosis than wild-type counterparts. Distinct Th2 or putative Th17 subsets expressing CCR4 or CCR10 accumulated more rapidly in Rgs16(-/-) lungs following challenge and produced proinflammatory cytokines IL-13 and IL-17B. CCR4(+)Rgs16(-/-) Th2 cells migrated excessively to CCL17 and localized aberrantly in challenged lungs. T lymphocytes were partially excluded from lung granulomas in Rgs16(-/-) mice, instead forming peribronchial/perivascular aggregates. Thus, RGS16-mediated confinement of T cells to Schistosome granulomas mitigates widespread cytokine-mediated pulmonary inflammation.

Accelerated and progressive and lethal liver fibrosis in mice that lack interleukin (IL)-10, IL-12p40, and IL-13Rα2.

BACKGROUND & AIMS: Progressive fibrosis contributes to the morbidity of several chronic diseases; it typically develops slowly, so the mechanisms that control its progression and resolution have been difficult to model. The proteins interleukin (IL)-10, IL-12p40, and IL-13Rα2 regulate hepatic fibrosis following infection with the helminth parasite Schistosoma mansoni. We examined whether these mediators interact to slow the progression of hepatic fibrosis in mice with schistosomiasis. METHODS: IL-10(-/-), IL-12/23(p40)(-/-), and IL-13Rα2(-/-) mice were crossed to generate triple knockout (TKO) mice. We studied these mice to determine whether the simultaneous deletion of these 3 negative regulators of the immune response accelerated mortality from liver fibrosis following infection with S mansoni. RESULTS: Induction of inflammation by S mansoni, liver fibrosis, and mortality increased greatly in TKO mice compared with wild-type mice; 100% of the TKO mice died by 10 weeks after infection. Morbidity and mortality were associated with the development of portal hypertension, hepatosplenomegaly, gastrointestinal bleeding, ascites, thrombocytopenia, esophageal and gastric varices, anemia, and increased levels of liver enzymes, all features of advanced liver disease. IL-10, IL-12p40, and IL-13Rα2 reduced the production and activity of the profibrotic cytokine IL-13. A neutralizing antibody against IL-13 reduced the morbidity and mortality of the TKO mice following S mansoni infection. CONCLUSIONS: IL-10, IL-12p40, and IL-13Rα2 act cooperatively to suppress liver fibrosis in mice following infection with S mansoni. This model rapidly reproduces many of the complications observed in patients with advanced cirrhosis, so it might be used to evaluate the efficacy of antifibrotic reagents being developed for schistosomiasis or other fibrotic diseases associated with a T-helper 2 cell-mediated immune response.

Colitis and intestinal inflammation in IL10-/- mice results from IL-13Rα2-mediated attenuation of IL-13 activity.

BACKGROUND & AIMS: The cytokine interleukin (IL)-10 is required to maintain immune homeostasis in the gastrointestinal tract. IL-10 null mice spontaneously develop colitis or are more susceptible to induction of colitis by infections, drugs, and autoimmune reactions. IL-13 regulates inflammatory conditions; its activity might be compromised by the IL-13 decoy receptor (IL-13Rα2). METHODS: We examined the roles of IL-13 and IL-13Rα2 in intestinal inflammation in mice. To study the function of IL-13Rα2, il10(-/-) mice were crossed with il13rα2(-/-) to generate il10(-/-)il13rα2(-/-) double knockout (dKO) mice. Colitis was induced with the gastrointestinal toxin piroxicam or Trichuris muris infection. RESULTS: Induction of colitis by interferon (IFN)-γ or IL-17 in IL-10 null mice requires IL-13Rα2. Following exposure of il10(-/-) mice to piroxicam or infection with T muris, production of IL-13Rα2 increased, resulting in decreased IL-13 bioactivity and increased inflammation in response to IFN-γ or IL-17A. In contrast to il10(-/-) mice, dKO mice were resistant to piroxicam-induced colitis; they also developed less severe colitis during chronic infection with T muris infection. In both models, resistance to IFN-γ and IL-17-mediated intestinal inflammation was associated with increased IL-13 activity. Susceptibility to colitis was restored when the dKO mice were injected with monoclonal antibodies against IL-13, confirming its protective role. CONCLUSIONS: Colitis and intestinal inflammation in IL10(-/-) mice results from IL-13Rα2-mediated attenuation of IL-13 activity. In the absence of IL-13Rα2, IL-13 suppresses proinflammatory Th1 and Th17 responses. Reagents that block the IL-13 decoy receptor IL-13Rα2 might be developed for inflammatory bowel disease associated with increased levels of IFN-γ and IL-17.

Mapping mouse IL-13 binding regions using structure modeling, molecular docking, and high-density peptide microarray analysis.

Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation, and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13Rα1 and IL-4Rα, which is also used by IL-4. IL-13 also binds to IL-13Rα2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13Rα2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling, and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13Rα1 and IL-13Rα2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13Rα2. In addition, the identification of peptide sequences that are unique to IL-13Rα1 allowed us to generate a monoclonal antibody that selectively binds IL-13Rα1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine-receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.