Caveolin1 interacts with the glucocorticoid receptor in the lung but is dispensable for its anti-inflammatory actions in lung inflammation and Trichuris Muris infection.

Glucocorticoids (Gcs) are widely prescribed anti-inflammatory compounds, which act through the glucocorticoid receptor (GR). Using an unbiased proteomics screen in lung tissue, we identified the membrane protein caveolin -1 (Cav1) as a direct interaction partner of the GR. In Cav1 knockout mice GR transactivates anti-inflammatory genes, including Dusp1, more than in controls. We therefore determined the role of Cav1 in modulating Gc action in two models of pulmonary inflammation. We first tested innate responses in lung. Loss of Cav1 impaired the inflammatory response to nebulized LPS, increasing cytokine/chemokine secretion from lung, but impairing neutrophil infiltration. Despite these changes to the inflammatory response, there was no Cav1 effect on anti-inflammatory capacity of Gcs. We also tested GR/Cav1 crosstalk in a model of allergic airway inflammation. Cav1 had a very mild effect on the inflammatory response, but no effect on the Gc response - with comparable immune cell infiltrate (macrophage, eosinophils, neutrophils), pathological score and PAS positive cells observed between both genotypes. Pursuing the Th2 adaptive immune response further we demonstrate that Cav1 knockout mice retained their ability to expel the intestinal nematode parasite T.muris, which requires adaptive Th2 immune response for elimination. Therefore, Cav1 regulates innate immune responses in the lung, but does not have an effect on Th2-mediated adaptive immunity in lung or gut. Although we demonstrate that Cav1 regulates GR transactivation of anti-inflammatory genes, this does not translate to an effect on suppression of inflammation in vivo.

IL-10R blockade during chronic schistosomiasis mansoni results in the loss of B cells from the liver and the development of severe pulmonary disease.

In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.

Migrating Schistosoma japonicum schistosomula induce an innate immune response and wound healing in the murine lung.

The migrating schistosomulum is an important stage of the schistosome lifecycle and represents a key target for elimination of infection by natural and vaccine-induced host immune responses. To gain a better understanding of how schistosomes initiate a primary host immune response we have characterised the host lung response to migrating Schistosoma japonicum schistosomula using a combination of histopathology, microarray analysis and real-time PCR. Our findings indicate that the early pulmonary response to these migrating larvae is characteristic of innate inflammation and wound healing. This response is associated with significant up-regulation of several genes with immunoregulatory function including Ch25h, Hmox1 and Retnla which may act to control the nature or magnitude of the inflammatory response to the migrating schistosomula, promoting both parasite and host survival. These findings contribute to our understanding of host-parasite interactions associated with schistosome and, especially, S. japonicum infection, and may aid the future design of novel vaccines that target the lung stage schistosomulum.

Basophils are the major producers of IL-4 during primary helminth infection.

IL-4 production by leukocytes is a key regulatory event that occurs early in the type 2 immune response, which induces allergic reactions and mediates expulsion of parasites. CD4(+) T cells and basophils are thought to be the key cell types that produce IL-4 during a type 2 response. In this study, we assessed the relative contribution of both CD4(+) T cell- and basophil-IL-4 production during primary and secondary responses to Nippostrongylus brasiliensis using a murine IL-4-enhanced GFP reporter system. During infection, IL-4-producing basophils were detected systemically, and tissue recruitment occurred independent of IL-4/STAT6 signaling. We observed that basophil recruitment to a tissue environment was required for their full activation. Basophil induction in response to secondary infection exhibited accelerated kinetics in comparison with primary infection. However, total basophil numbers were not enhanced, as predicted by previous models of protective immunity. Overall, the induction and migration of IL-4-producing basophils into peripheral tissues was found to be a prominent characteristic of the primary but not memory responses to N. brasiliensis infection, in which CD4(+) T cells were identified as the major source of IL-4. Whereas basophils were the major initial producers of IL-4, we determined that normal Th2 differentiation occurs independently of basophils, and depletion of basophils led to an enhancement of inflammatory cell recruitment to the site of infection.

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses.

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.

Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of a chronic pulmonary fungal infection accompanied by an "allergic" response (T2) to the infection, i.e., a model of an allergic bronchopulmonary mycosis. Our objective was to determine whether IFN-gamma plays a role in regulating the pulmonary T2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in IFN-gamma knockout (KO) mice, resulting in an increased pulmonary burden of fungi at wk 3. IFN-gamma was required for the early influx of leukocytes into the lungs but was not required later in the infection. By wk 3, eosinophil and macrophage numbers were elevated in the absence of IFN-gamma. The inducible NO synthase to arginase ratio was lower in the lungs of IFN-gamma KO mice and the macrophages had increased numbers of intracellular cryptococci and YM1 crystals, indicative of alternatively activated macrophages in these mice. There was evidence of pulmonary fibrosis in both wild-type and IFN-gamma KO mice by 5 wk postinfection. IFN-gamma production was not required for the development of T2 cytokine (IL-4, IL-5, IL-13) producing cells in the lungs and lung-associated lymph nodes or induction of an IgE response. At a number of time points, T2 cytokine production was enhanced in IFN-gamma KO mice. Thus, in the absence of IFN-gamma, C57BL/6 mice develop an augmented allergic response to C. neoformans, including enhanced generation of alternatively activated macrophages, which is accompanied by a switch from a chronic to a progressive pulmonary cryptococcal infection.

Pulmonary dirofilariasis with serologic study on familial infection with Dirofilaria immitis.

An asymptomatic patient with a pulmonary coin lesion surgically diagnosed with pulmonary dirofilariasis caused by infection with Dirofilaria immitis (D. immitis) is presented. The preoperative stored serum of the patient was positive for D. immitis by enzyme-linked immunosorbent assay (ELISA). A family study showed that three of five family members were seropositive for D. immitis. These results suggest that family members of a patient with pulmonary dirofilariasis were frequently exposed to D. immitis and serodiagnostic methods are useful for detecting subclinical infection of D. immitis.

Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair.

T helper 1 responses are typically proinflammatory, while Th2 responses have been considered regulatory. Interestingly, Th2 responses characterize a number of pulmonary diseases, many of which terminate in tissue remodeling and fibrosis. We developed a mouse model using Schistosoma mansoni eggs and cytokine-deficient mice to induce highly polarized Th1- or Th2-type inflammation in the lung. In this study, we examined the pathology and cytokine profiles in Th1- and Th2-polarized environments and used oligonucleotide microarray analysis to decipher the genes responsible for these effects. We further elaborated on the results using IL-10- and IL-13-deficient mice because these cytokines are believed to be the central regulators of Th2-associated pathology. We found that the Th1-polarized mice developed small granulomas with less fibrosis while expressing genes characteristic of tissue damage. Th2-polarized mice, in contrast, formed large granulomas with massive collagen deposition and up-regulated genes associated with wound healing, specifically, arginase, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of MMP. In addition, several members of the chitinase-like family were up-regulated in the lung following egg challenge. We also developed a method of defining the net collagen deposition using the expression profiles of several collagen, MMP, and tissue inhibitors of MMP genes. We found that Th1-polarized mice did not elaborate collagens or MMPs and therefore did not have a significant capacity for repair in this model. Thus, Th1-mediated inflammation is characterized by tissue damage, while Th2 directs wound healing and fibrosis.

Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.

Type 2-biased expression of cytokine genes in lung granulomatous lesions induced by Nippostrongylus brasiliensis infection.

Infections with helminthic parasites occasionally induce pulmonary diseases with possible involvement of immunological mechanisms. In rats infected with the nematode Nippostrongylus brasiliensis, pulmonary granulomatous lesions develop and persist after the larvae have migrated through the lungs. To determine the pathogenesis of this lesion, we examined cytokine gene expression in the lungs using RT-PCR and in situ hybridization. Two weeks after infection, when fully developed lesions appeared, levels of IL-3 and of type2 cytokines IL-4, IL-5, IL-6 and IL-13 gene expression were markedly enhanced in whole lung homogenates. Those of IL-2 and IFN-gamma were also slightly increased 2 weeks postinfection. IL-12 mRNA level did not change after 2 weeks but was slightly increased after 4 weeks. Levels of IL-10 and proinflammatory cytokine TNF gene expression did not show significant changes, although a slight increase was observed in IL-1beta message after 2 weeks. In situ hybridization studies showed that lung granulomatous lesions were composed mainly of lymphoid cells expressing IL-3, IL-4 and IL-13 mRNA, but not IFN-gamma mRNA. IL-5 mRNA-expressing cells were fewer in number than these cells. RMCP II immunohistochemistry revealed that mast cells increased in number in the lung granulomas. From these results, it was concluded that the nematode infection-associated lung granuloma was a type 2 lesion.

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.

Changes in cytokine levels during reactivation of Toxoplasma gondii infection in lungs.

We studied cytokine proteins and mRNAs in mice with two forms of Toxoplasma gondii pneumonia resulting from reactivation of infection. In the first form, mice were infected with T. gondii, developed and recovered from systemic disease, and then developed pneumonia 3 weeks later. As pulmonary inflammation developed, levels of cytokine mRNAs for gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 increased in bronchoalveolar lavage (BAL) cells or lung tissue, and the level of IFN-gamma protein increased in BAL fluid. The second form of pneumonia occurred as a complication of primary cytomegalovirus (CMV) disease in mice with dormant T. gondii infection. During CMV disease, IL-2 mRNA levels decreased in lung tissue, IL-10 protein levels increased in lung tissue, and IL-10 protein levels increased in BAL fluid. As the mice recovered from CMV disease, T. gondii infection was reactivated in the lungs and was manifested as T. gondii pneumonia. During CMV-induced T. gondii pneumonia, IFN-gamma, IL-2, IL-4, and IL-10 mRNA levels increased in BAL cells or lung tissue, and both IFN-gamma and IL-2 protein levels increased in BAL fluid. We concluded that both forms of T. gondii pneumonia are accompanied by increases in both type 1 T-helper and type 2 T-helper cytokine levels in lungs. The mechanism of CMV-induced reactivation of T. gondii infection in lungs may involve local decreases in IL-2 levels and/or increases in IL-10 levels.

Analysis of granuloma formation in double cytokine-deficient mice reveals a central role for IL-10 in polarizing both T helper cell 1- and T helper cell 2-type cytokine responses in vivo.

In response to i.v.-injected eggs of Schistosoma mansoni, normal mice develop a dominant type 2 response, whereas IL-10-deficient animals generate a mixed type 1/type 2 cytokine profile and show reduced pulmonary granuloma formation. IL-4-deficient mice, while displaying diminished type 2 responses and granulomatous inflammation, also do not fully default to a type 1 cytokine profile. Strikingly, mice doubly deficient in IL-4 and IL-10 are completely defective in pulmonary granuloma formation and develop a highly polarized type 1 cytokine pattern. In analogous fashion, mice deficient in both IL-12 and IL-10 generate highly exacerbated type 2 cytokine responses, whereas in wild-type animals, IL-12 depletion minimally effects egg-induced cytokine production. Together, these results argue first that IL-10 is an important endogenous down-regulator of type 2 as well as type 1 cytokine synthesis, and second, that its induction is critical for type 2 response polarization in vivo.