Mara G. Shainheit

IL-23 Is Required for the Development of Severe Egg-Induced Immunopathology in Schistosomiasis and for Lesional Expression of IL-17

In infection with the trematode helminth Schistosoma mansoni, the severity of CD4 T cell-mediated hepatic granulomatous and fibrosing inflammation against parasite eggs varies considerably in humans and among mouse strains. In mice, either the natural high pathology, or high pathology induced by concomitant immunization with schistosome egg Ags (SEA) in CFA (SEA/CFA), results from a failure to contain a net proinflammatory cytokine environment. We previously demonstrated that the induction of severe immunopathology was dependent on the IL-12/IL-23 common p40 subunit, and correlated with an increase in IL-17, thus implying IL-23 in the pathogenesis. We now show that mice lacking the IL-23-specific subunit p19 are impaired in developing severe immunopathology following immunization with SEA/CFA, which is associated with a marked drop of IL-17 in the granulomas, but not in the draining mesenteric lymph nodes, and with a markedly suppressed SEA-specific IFN-γ response regulated by a striking increase in IL-10. The granulomas are characterized by a significant reduction in Gr-1+ cell recruitment and by alternative macrophage activation. Taken together, these results demonstrate that IL-23 per se is not necessary for the generation of IL-17-producing T cells, but is essential for the development of severe schistosome egg-induced immunopathology, and its absence cannot be overcome with other possible compensatory mechanisms.

Dendritic Cell IL-23 and IL-1 Production in Response to Schistosome Eggs Induces Th17 Cells in a Mouse Strain Prone to Severe Immunopathology

Infection with schistosomes results in a CD4 T cell-mediated inflammatory reaction against parasite eggs that varies greatly in magnitude both in humans as well as in mice. In the murine disease, the severe form of immunopathology correlates with high levels of IL-17. We now report that live schistosome eggs stimulate dendritic cells from high pathology-prone CBA mice to produce IL-12p40, IL-6, and TGF-β, whereas those from low pathology-prone BL/6 mice only make TGF-β. Moreover, egg-stimulated dendritic cells plus naive CD4 T cells from CBA mice resulted in increased levels of IL-6, IL-23, IL-1β, as well as IL-17 and the chemokines CXCL1, CXCL2, and CCL2, whereas similarly treated BL/6 cell cocultures instead expressed higher IL-4, IL-5, IL-10, and the transcription factor Foxp3. Neutralization of IL-23 and IL-1, but not of IL-6 or IL-21, profoundly inhibited egg-induced IL-17 production in the CBA cocultures. Conversely, stimulation with schistosome eggs in the presence of exogenous IL-23 and IL-1β induced BL/6 cells to make IL-17. These findings identify IL-23 and IL-1 as critical host factors that drive IL-17 production, and suggest that parasite recognition followed by a genetically determined innate proinflammatory response induces the development of Th17 cells and thus controls the outcome of immunopathology in schistosomiasis.

Coinfection with the Intestinal Nematode Heligmosomoides polygyrus Markedly Reduces Hepatic Egg-Induced Immunopathology and Proinflammatory Cytokines in Mouse Models of Severe Schistosomiasis

ABSTRACT Infection with the trematode helminth Schistosoma mansoni results in a parasite egg-induced, CD4 T-cell-mediated, hepatointestinal granulomatous and fibrosing inflammation that varies greatly in severity, with a higher frequency of milder forms typically occurring in regions where the disease is endemic. One possible explanation for this is that in these regions the degree of inflammation is lessened by widespread concurrent infection with gastrointestinal nematodes. We tested this hypothesis by establishing a murine coinfection model in which mice were infected with the intestinal nematode parasite Heligmosomoides polygyrus prior to infection with S. mansoni. In CBA mice that naturally display a severe form of schistosomiasis, preinfection with H. polygyrus resulted in a marked reduction in schistosome egg-induced hepatic immunopathology, which was associated with significant decreases in the levels of interleukin-17 (IL-17), gamma interferon, tumor necrosis factor alpha, IL-23, IL-6, and IL-1β and with increases in the levels of IL-4, IL-5, IL-10, and transforming growth factor β in mesenteric lymph node cells, purified CD4 T cells, and isolated liver granuloma cells. There also were increases in liver Ym1 and forkhead box P3 transcription factor expression. In another model of high-pathology schistosomiasis induced in C57BL/6 mice by immunization with schistosome egg antigens in complete Freunds adjuvant, coinfection with the nematodes also resulted in a marked inhibition of hepatic immunopathology accompanied by similar shifts in cytokine production. These findings demonstrate that intestinal nematodes prevent Th1- and Th17-cell-mediated inflammation by promoting a strong Th2-polarized environment associated with increases in the levels of alternatively activated macrophages and T regulatory cells, which result in significant amelioration of schistosome-induced immunopathology.

Induction and regulation of pathogenic Th17 cell responses in schistosomiasis

Schistosomiasis is a major tropical disease caused by trematode helminths in which the host mounts a pathogenic immune response against tissue-trapped parasite eggs. The immunopathology consists of egg antigen-specific CD4 T cell-mediated granulomatous inflammation that varies greatly in magnitude in humans and among mouse strains in an experimental model. New evidence, covered in this review, intimately ties the development of severe pathology to IL-17-producing CD4 T helper (Th17) cells, a finding that adds a new dimension to the traditional CD4 Th1 vs. Th2 cell paradigm. Most examined mouse strains, in fact, develop severe immunopathology with substantial Th17 as well as Th1 and Th2 cell responses; a solely Th2-polarized response is an exception that is only observed in low-pathology strains such as the C57BL/6. The ability to mount pathogenic Th17 cell responses is genetically determined and depends on the production of IL-23 and IL-1β by antigen presenting cells following recognition of egg antigens; analyses of several F2 progenies of (high × low)-pathology strain crosses demonstrated that quantitative trait loci governing IL-17 levels and disease severity vary substantially from cross to cross. Low pathology is dominant, which may explain the low incidence of severe disease in humans; however, coinfection with intestinal nematodes can also dampen pathogenic Th17 cell responses by promoting regulatory mechanisms such as those afforded by alternatively activated macrophages and T regulatory cells. A better understanding of the pathways conducive to severe forms of schistosomiasis and their regulation should lead to interventions similar to those presently used to manage other immune-mediated diseases.

The Pathogenic Th17 Cell Response to Major Schistosome Egg Antigen Is Sequentially Dependent on IL-23 and IL-1β

CBA/J mice infected with the helminth Schistosoma mansoni develop severe CD4 T cell-mediated hepatic granulomatous inflammation against parasite eggs associated with a robust Th17 cell response. We investigated the requisites for Th17 cell development using novel CD4 T cells expressing a transgenic TCR specific for the major Sm-p40 egg Ag, which produce IL-17 when stimulated with live schistosome eggs. Neutralization of IL-23 or blockade of the IL-1 receptor, but not IL-6 neutralization, abrogated egg-induced IL-17 secretion by transgenic T cells, whereas exogenous IL-23 or IL-1β reconstituted their ability to produce IL-17 when stimulated by syngeneic IL-12p40–deficient dendritic cells. Kinetic analysis demonstrated that IL-17 production was initiated by IL-23 and amplified by IL-1β. Significantly, schistosome-infected IL-12p40–deficient or IL-1R antagonist-treated CBA/J mice developed markedly reduced hepatic immunopathology with a dampened egg Ag-specific IL-17 response. These results demonstrate that the IL-23–IL-1–IL-17 axis has a central role in the development of severe schistosome egg-induced immunopathology.

The core promoter of the capsule operon of Streptococcus pneumoniae is necessary for colonization and invasive disease.

ABSTRACT Streptococcus pneumoniae is a commensal of the human nasopharynx but can cause invasive diseases, including otitis media, pneumonia, sepsis, and meningitis. The capsular polysaccharide (capsule) is a critical virulence factor required for both asymptomatic colonization and invasive disease, yet the expression level is different in each anatomical site. During colonization, reduced levels of capsule promote binding to the host epithelium and biofilm formation, while during systemic infection, increased capsule is required to evade opsonophagocytosis. How this regulation of capsule expression occurs is incompletely understood. To investigate the contribution of transcriptional regulation on capsule level in the serotype 4 strain TIGR4, we constructed two mutants harboring a constitutive promoter that was either comparably weaker (P cat ) or stronger (P tRNAGlu ) than the wild-type (WT) capsule promoter, P cps . Mild reductions in cpsA and cpsE transcript levels in the P cat promoter mutant resulted in a 2-fold reduction in total amounts of capsule and in avirulence in murine models of lung and blood infection. Additionally, the P tRNAGlu mutant revealed that, despite expressing enhanced levels of cpsA and cpsE and possessing levels of capsule comparable to those of WT TIGR4, it was still significantly attenuated in all tested in vivo niches. Further analysis using chimeric promoter mutants revealed that the WT −10 and −35 boxes are required for optimal nasopharyngeal colonization and virulence. These data support the hypothesis that dynamic transcriptional regulation of the capsule operon is required and that the core promoter region plays a central role in fine-tuning levels of capsule to promote colonization and invasive disease.

Regulated assembly of vacuolar ATPase is increased during cluster disruption-induced maturation of dendritic cells through a phosphatidylinositol 3-kinase/mTOR-dependent pathway.

Background: The V-ATPase is induced to assemble during TLR-activated maturation of dendritic cells. Results: Cluster disruption of dendritic cells also induces V-ATPase assembly by a PI3K/mTOR-dependent mechanism. Conclusion: Semi-mature dendritic cells show increased V-ATPase assembly. Significance: Dendritic cell maturation associated with immune tolerance involves V-ATPase assembly. The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.

Genetic Control of Severe Egg-Induced Immunopathology and IL-17 Production in Murine Schistosomiasis

Infection with the trematode parasite Schistosoma mansoni results in a distinct heterogeneity of disease severity, both in humans and in an experimental mouse model. Severe disease is characterized by pronounced hepatic egg-induced granulomatous inflammation in a proinflammatory cytokine environment, whereas mild disease corresponds with reduced hepatic inflammation in a Th2 skewed cytokine environment. This marked heterogeneity indicates that genetic differences play a significant role in disease development, yet little is known about the genetic basis of dissimilar immunopathology. To investigate the role of genetic susceptibility in murine schistosomiasis, quantitative trait loci analysis was performed on F2 progeny derived from SJL/J and C57BL/6 mice, which develop severe and mild pathology, respectively. In this study, we show that severe liver pathology in F2 mice 7 wk after infection significantly correlated with an increase in the production of the proinflammatory cytokines IL-17, IFN-γ, and TNF-α by schistosome egg Ag-stimulated mesenteric lymph node cells. Quantitative trait loci analysis identified several genetic intervals controlling immunopathology as well as IL-17 and IFN-γ production. Egg granuloma size exhibited significant linkage to two loci, D4Mit203 and D17Mit82, both of which were inherited in a BL/6 dominant manner. Furthermore, a significant reduction of hepatic granulomatous inflammation and IL-17 production in interval-specific congenic mice demonstrated that the two identified genetic loci have a decisive effect on the development of immunopathology in murine schistosomiasis.

CD209a Expression on Dendritic Cells Is Critical for the Development of Pathogenic Th17 Cell Responses in Murine Schistosomiasis

In murine schistosomiasis, immunopathology and cytokine production in response to parasite eggs are uneven and strain dependent. CBA/J (CBA) mice develop severe hepatic granulomatous inflammation associated with prominent Th17 cell responses driven by dendritic cell (DC)-derived IL-1β and IL-23. Such Th17 cells fail to develop in low-pathology C57BL/6 (BL/6) mice, and the reasons for these strain-specific differences in APC reactivity to eggs remain unclear. We show by gene profiling that CBA DCs display an 18-fold higher expression of the C-type lectin receptor CD209a, a murine homolog of human DC-specific ICAM-3–grabbing nonintegrin, compared with BL/6 DCs. Higher CD209a expression was observed in CBA splenic and granuloma APC subpopulations, but only DCs induced Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DCs and overexpression in BL/6 DCs demonstrated that CD209a is essential for egg-elicited IL-1β and IL-23 production and subsequent Th17 cell development, which is associated with SRC, RAF-1, and ERK1/2 activation. These findings reveal a novel mechanism controlling the development of Th17 cell–mediated severe immunopathology in helminthic disease.

Disruption of Interleukin-27 Signaling Results in Impaired Gamma Interferon Production but Does Not Significantly Affect Immunopathology in Murine Schistosome Infection

ABSTRACT In schistosomiasis mansoni, parasite eggs cause hepatointestinal granulomatous inflammation and fibrosis mediated by CD4 T cells specific for egg antigens. The severity of disease varies extensively in humans and among mouse strains. Marked disease exacerbation induced in typically low-pathology C57BL/6 mice by immunization with schistosome egg antigens (SEA) in complete Freunds adjuvant (SEA/CFA) correlates with elevated production of the proinflammatory cytokines gamma interferon (IFN-γ) and interleukin-17 (IL-17), which are regulated by IL-12 and IL-23, respectively. Here we examined the effect on the schistosome infection of a third member of the IL-12 family of heterodimeric cytokines, IL-27, using SEA/CFA-immunized and unimmunized mice deficient in the IL-27 receptor chain WSX-1 (WSX-1−/−). SEA-stimulated bulk mesenteric lymph node cells or CD4 T cells from 7-week-infected WSX-1−/− mice produced significantly less IFN-γ than did those from C57BL/6 mice, even though there was no difference between these mice in exacerbated hepatic egg-induced granulomatous inflammation or in the levels of IL-17 induced by immunization with SEA/CFA. A fraction of the cells in the granulomas stained positive for IL-27, but there were no significant differences between WSX-1−/− and BL/6 mice, nor were there differences in the number of CD4 T cells and eosinophils. A 24-week chronic infection resulted in markedly reduced levels of proinflammatory cytokines, including IFN-γ, in WSX-1−/− mice, but again the magnitude of immunopathology was not significantly different between the two groups. These findings indicate that despite the impaired IFN-γ production, IL-27 signaling has no significant effect on either the magnitude of egg-induced immunopathology or on its closest in vitro correlate, IL-17.