Philip Smith

IL-9-Deficient Mice Establish Fundamental Roles for IL-9 in Pulmonary Mastocytosis and Goblet Cell Hyperplasia but Not T Cell Development

Interleukin-9 is a cytokine produced by Th2 cells and is a candidate gene for asthma and atopy. We have generated IL-9-deficient mice to delineate the specific roles of IL-9 in Th2 responses. Using a pulmonary granuloma model, we have demonstrated a distinct requirement for IL-9 in the rapid and robust generation of pulmonary goblet cell hyperplasia and mastocytosis in response to lung challenge. In contrast, eosinophilia and granuloma formation were not affected. IL-9 was not required for T cell development or differentiation, the generation of naive or antigen-driven antibody responses, or the expulsion of the intestinal parasitic nematode Nippostrongylus brasiliensis. Thus, deletion of IL-9 manifests as a highly defined phenotype in Th2 responses modulating mucus production and mast cell proliferation.

Schistosoma mansoni secretes a chemokine binding protein with antiinflammatory activity

The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the hosts innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been selected to modulate the host to tolerate chronic worm infections, often for decades, without overt morbidity. The modulation of immunity by schistosomes has been shown to prevent a range of immune-mediated diseases, including allergies and autoimmunity. Individual immune-modulating schistosome molecules have, therefore, therapeutic potential as selective manipulators of the immune system to prevent unrelated diseases. Here we show that S. mansoni eggs secrete a protein into host tissues that binds certain chemokines and inhibits their interaction with host chemokine receptors and their biological activity. The purified recombinant S. mansoni chemokine binding protein (smCKBP) suppressed inflammation in several disease models. smCKBP is unrelated to host proteins and is the first described chemokine binding protein encoded by a pathogenic human parasite and may have potential as an antiinflammatory agent.

Inhibition of Type 1 Cytokine–mediated Inflammation by a Soluble CD30 Homologue Encoded by Ectromelia (Mousepox) Virus

CD30 is up-regulated in several human diseases and viral infections but its role in immune regulation is poorly understood. Here, we report the expression of a functional soluble CD30 homologue, viral CD30 (vCD30), encoded by ectromelia (mousepox) virus, a poxvirus that causes a severe disease related to human smallpox. We show that vCD30 is a 12-kD secreted protein that not only binds CD30L with high affinity and prevents its interaction with CD30, but it also induces reverse signaling in cells expressing CD30L. vCD30 blocked the generation of interferon γ–producing cells in vitro and was a potent inhibitor of T helper cell (Th)1- but not Th2-mediated inflammation in vivo. The finding of a CD30 homologue encoded by ectromelia virus suggests a role for CD30 in antiviral defense. Characterization of the immunological properties of vCD30 has uncovered a role of CD30–CD30L interactions in the generation of inflammatory responses.

IL-13 Overexpression Predisposes to Anaphylaxis Following Antigen Sensitization

Anaphylaxis represents an extreme form of allergic reaction. This acute-phase component of allergy and asthma is triggered by allergen-induced degranulation of mast cells following the cross-linking of cell surface-bound, allergen-specific IgE, resulting in the liberation of inflammatory mediators and the development of bronchoconstriction. We used IL-13 transgenic mice to investigate the role of this Th2 cell-derived cytokine in the onset of allergic disease. Strikingly, IL-13-transgenic mice were highly predisposed to fatal anaphylaxis following Ag sensitization. This response correlated with substantially elevated levels of circulating Ag-specific IgE, mast cell degranulation, and histamine release. Furthermore, allergen exposure also induced phenotypic changes typical of asthma, including pulmonary fibrosis, goblet cell hyperplasia, elevated Th2 cytokines, eosinophilia, and airways occluded by mucus and Charcot-Leyden crystals. Expression of IL-4 was not required for the induction of IgE-mediated responses. These data represent the first characterization of a functional role for IL-13-induced IgE in the generation of immediate hypersensitivity reactions and highlight the importance of IL-13 in the development of the symptoms of atopy. The systemic regulation of this response makes these mice an important resource for studying atopic responses.

Type 1 and type 2 cytokine-producing mouse CD4+ and CD8+ T cells in acute Schistosoma mansoni infection.

CD4+ and CD8+ T cells can be divided based on the cytokines that they secrete into type 1 (Th1, Tc1) and type 2 (Th2, Tc2) subsets. Schistosoma mansoni infection in mice is characterized by a type 2‐dominated response. We have used intracellular cytokine staining to demonstrate dramatic changes in the relative numbers of Tc1 and Th2 cells in the spleens of mice during acute schistosome infection. In infected mice prior to egg laying a generalized type 1 response dominated, and was associated with an expansion in the frequency of Tc1 and Th1 cells. By week 7 after infection the cytokine response was of type 2, with an increase in the numbers of Th2 cells and a dramatic reduction in the frequency of Tc1 cells. Following the onset of egg laying there was apoptosis of cells in the spleens of mice, with CD4+ and in particular CD8+ T cells undergoing apoptosis. The loss of CD8+ T cells may in part be attributable to the development of a type 2 environment, following egg laying, with type 2 responses mediating the apoptosis of Tc1 cells. Schistosome regulation of Tc1 during egg laying may be required to prevent type 1 inflammatory responses from exacerbating egg‐induced pathology.

Elevated type 1, diminished type 2 cytokines and impaired antibody response are associated with hepatotoxicity and mortalities during Schistosoma mansoni infection of CD4‐depleted mice

During murine Schistosoma mansoni infections parasite eggs evoke a type 2 cytokine‐dependent and CD4+ T cell‐mediated granulomatous response in the liver. In this study CD4+ T cell‐depleted CBA / Ca mice developed hepatic steatosis and had high mortalities during early acute schistosome infection. CD4‐depleted mice had smaller liver granulomas and reduced hepatic fibrosis. The hepatocytotoxicity was characterized by microvesicular steatosis and neutrophil infiltration. The livers of depleted mice had similar levels of apoptosis as control infected mice but had a marked increase in lipid peroxidation indicative of their livers being under oxidative stress. CD4‐depleted mice had impaired egg excretion and exacerbated intestinal pathology. A type 1 cytokine‐dominated response was present in infected CD4‐depleted mice and relatively reduced production of type 2 cytokines. Antibody responses to parasite antigens were also substantially reduced. Transfer of immune serum or IgG significantly delayed mortalities in depleted mice and prevented hepatocyte damage. Although biasing the cytokine dichotomy to a type 1‐dominated response during murine schistosome infection is desirable with respect to certain pathological processes, i.  e. it will reduce the granulomatous inflammation and hepatic fibrosis, these effects contribute to fatal pathology if there is reduced protective type 2 cytokines and a defect in antibody responses.

Defective in vivo induction of functional type 2 cytokine responses in aged mice

Aged mice have various defects in their immune system. We report that following in vivo challenge with type 2 cytokine‐inducing Schistosoma mansoni eggs, aged mice fail to produce type 2 cytokines and also have impaired antigen‐specific antibody production. Using two separate type 2 cytokine‐dependent in vivo models, the synchronous pulmonary schistosome egg granuloma model and infection with the gastro‐intestinal nematode Nippostrongylus brasiliensis, aged mice were shown to have a dramatically impaired capacity to elicit a functional type 2 response, i. e. respectively, impaired pulmonary granulomas and delayed rejection of intestinal worms. Aged mice did not develop eosinophilia and had impaired production of antigen specific IgE. Defective induction of type 2 responses was associated with negligible IL‐2 and elevated IFN‐γ production by cells from aged mice. Naive aged mice had increased numbers of Th1, Th2 and Tc1 cells compared to young animals. In vivo type 2 challenge increased the frequencies of Th1 and Tc1 cells and reducing Th2 cell numbers in aged mice. These data demonstrate that a consequence of ageing is a profound in vivo defect in the capacity to elicit type 2 cytokine responses and such an impairment in type 2 responsiveness may account for the increased incidence of various type 1 cytokine‐mediated diseases in aged individuals.

The C-type lectin SIGNR1 binds Schistosoma mansoni antigens in vitro, but SIGNR1-deficient mice have normal responses during schistosome infection.

ABSTRACT The de novo immune response to infectious organisms arises from the innate recognition of pathogen-associated molecular patterns (PAMPs) by the hosts pattern recognition receptors (PRRs). As the generation of type 2 cytokine responses by the human trematode parasite Schistosoma mansoni is glycan mediated, there is a particular potential role for a C-type lectin receptor (CLR) to mediate the innate recognition of schistosome PAMPs. One such CLR, dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209), has been shown to recognize glycans expressed by S. mansoni eggs. We show that SIGNR1 (SIGN-related 1; CD209b), a murine homologue of DC-SIGN that is expressed on macrophages, also binds both schistosome-soluble egg antigens and worm antigens in vitro. The generation of schistosome egg-induced pulmonary egg granulomas was not altered in SIGNR1-deficient mice. Following S. mansoni infection, the SIGNR1-deficient mice had an unaltered phenotype with an intact immunological response and no difference in pathology. In this study we demonstrate that although SIGNR1 recognizes S. mansoni antigens in vitro, this CLR is redundant during infection. This study highlights the finding that although there was binding of SIGNR1 to immunogenic factors produced in the S. mansoni life cycle, this recognition does not translate to a functional in vivo role for the PRR during infection.

Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization

Background: P2X7 receptors are important in inflammation and immunity, but excessive activation results in cell death. Results: Facilitation of P2X7 receptor currents and dilation of the pore are enhanced by cholesterol depletion and reduced by cholesterol loading. Conclusion: Plasma membrane cholesterol destabilizes the open and dilated states of the receptor pore. Significance: Targeting of P2X7 to cholesterol-rich lipid rafts may protect cells from death. P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na+ and N-methyl-d-glucamine (NMDG+) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.

SDHA related tumorigenesis: a new case series and literature review for variant interpretation and pathogenicity

To evaluate the role of germline SDHA mutation analysis by (1) comprehensive literature review, (2) description of novel germline SDHA mutations and (3) in silico structural prediction analysis of missense substitutions in SDHA.