Laura I. Rutitzky

Protective immune mechanisms in helminth infection

Important insights have recently been gained in our understanding of how host immune responses mediate resistance to parasitic helminths and control associated pathological responses. Although similar cells and cytokines are evoked in response to infection by helminths as diverse as nematodes and schistosomes, the components of the response that mediate protection are dependent on the particular parasite. In this Review, we examine recent findings regarding the mechanisms of protection in helminth infections that have been elucidated in murine models and discuss the implications of these findings in terms of future therapies.

Severe CD4 T Cell-Mediated Immunopathology in Murine Schistosomiasis Is Dependent on IL-12p40 and Correlates with High Levels of IL-17

C57BL/6 mice infected with the helminth Schistosoma mansoni develop small hepatic granulomas around parasite eggs, but concomitant immunization with soluble schistosome egg Ags (SEA) in CFA (SEA/CFA) causes marked exacerbation of the lesions in a Th1-dominated environment characterized by high levels of IFN-γ. We explored the cause of the severe immunopathology by using IL-12p40−/− and IL-12p35−/− mice. SEA/CFA-immunized IL-12p40−/− mice, incapable of making IL-12 or IL-23, were completely resistant to high pathology, and their SEA-stimulated lymphoid cells failed to secrete significant IFN-γ or IL-17. In contrast, SEA/CFA-immunized IL-12p35−/− mice, able to make IL-23 but not IL-12, developed severe lesions that correlated with high levels of IL-17, low IFN-γ, and an expansion of activated CD4 T cells with a CD44high/CD62Llow memory phenotype. In vivo administration of neutralizing anti-IL-17 mAb markedly inhibited hepatic granulomatous inflammation. Importantly, CBA mice, a naturally high pathology strain, also displayed elevated IL-17 levels comparable to those seen in the SEA/CFA-immunized BL/6 mice, and their lesions were similarly reduced by in vivo treatment with anti-IL-17. Our findings indicate that an IL-17-producing T cell population, likely driven by IL-23, significantly contributes to severe immunopathology in schistosomiasis.

IL-10 is crucial for the transition from acute to chronic disease state during infection of mice with Schistosoma mansoni

After infection of mice with Schistosoma mansoni, deposition of eggs in the walls of the intestine and liver provokes an intense (acute) T cell response that peaks at week 8 and, thereafter, down‐modulates as the disease becomes chronic. Egg antigen‐stimulated proliferation of mesenteric lymph node and spleen cells in vitro was intense at week 8 in both IL‐10–/– and wild‐type (WT) mice, while proliferative responses were markedly reduced at week 15 in WT mice, but undiminished in IL‐10–/– animals. Moreover, in the absence of IL‐10 production, levels of both IFN‐γ and IL‐4 remained elevated at week 15. Granulomas around eggs embolized in the livers of WT mice were significantly smaller at week 15 than week 8, whereas those in IL‐10–/ animals were larger at week 8, showed no reduction in size at week 15, and were less sharply demarcated by peripheral collagen. There was also a greater leukocytic infiltration and necrosis of the hepatic parenchyma. These data suggest that in mice IL‐10 regulates not only the intensity of hepatic inflammation, but also granuloma organization and cohesiveness. It is a crucial agent in the down‐modulation of immune responses and immunopathology that defines the transition from acute to chronic disease.

The immunobiology of Th1 polarization in high‐pathology schistosomiasis

Summary:  Schistosomiasis is a serious global helminthic disease, in which the main immunopathology consists of a granulomatous and fibrosing reaction against tissue‐trapped parasite eggs. The severity of this inflammatory process, the product of a CD4+ T‐cell‐mediated immune response against parasite egg antigens, is, however, markedly uneven, both in human patients and among mouse strains in an experimental model. Severe schistosomiasis is associated with persistently elevated pro‐inflammatory T‐helper‐1 (Th1)‐type cytokines, whereas milder pathology is present when Th2 cytokines dominate. This scenario is supported by the pronounced pathology resulting from the obliteration of pathways that facilitate Th2 differentiation and by the development of more intense lesions in mouse strains that fail to downregulate the Th1 response. Genetically prone high‐pathology mice have a higher proportion of CD4+ T cells in lymph nodes and granulomas, in which the Th1 phenotype is driven by interleukin‐12; they also develop a dominant repertoire against peptide 234–246 of the major Sm‐p40 egg antigen, utilizing a strikingly restricted T‐cell receptor structure that involves Vα11.3β8. In turn, low‐pathology mice exhibit enhanced CD4+ T‐cell apoptosis, which contributes to limit pathology. The definition of distinctive immune profiles associated with polar forms of schistosomiasis opens opportunities for targeted immuno‐intervention in individuals suffering from or at risk of severe disease.

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.

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity

Significance IgG fragment crystallizable domain (Fc) sialylation has emerged as an important but controversial concept for regulating anti-inflammatory activity of antibodies. Moreover, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for human use. We describe for the first time, to our knowledge, the development of a robust, scalable process to generate a sialylated intravenous immunoglobulin (IVIg) drug candidate with maximum Fc sialylation devoid of unwanted modifications. By using a wide panel of physicochemical analytics and in vivo models, we have validated the quality and potent anti-inflammatory activity of this clinical candidate. This report not only confirms the controversial anti-inflammatory activity of IgG-Fc sialylation, it also represents the first sialylated IVIg preparation, to our knowledge, with consistent anti-inflammatory potency suitable for clinical development. Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc–sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.

Th1-polarizing immunization with egg antigens correlates with severe exacerbation of immunopathology and death in schistosome infection

In schistosomiasis mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammatory process, which is mediated by, and dependent on, MHC class II-restricted CD4 T helper (Th) lymphocytes specific for schistosome egg antigens (SEA). In the mouse model of the disease, CBA mice develop large granulomas, whereas in C57BL/6 (BL/6) mice these granulomas are significantly smaller. To further investigate how the prevailing cytokine environment influences the development of the egg-induced immunopathology, we immunized the low-pathology BL/6 mice with SEA in complete Freunds adjuvant (CFA) once before, and once again during, the course of a 7-week infection. This immunization caused a pronounced Th1 shift in the SEA-specific CD4 T cell response, which was detected in the mesenteric lymph nodes (MLNs) and spleens, as well as in the granulomatous lesions themselves. The immunized mice displayed a dramatic enhancement of hepatic egg-induced immunopathology manifested by a marked increase in granuloma size and parenchymal inflammation, leading to early death. Control mice immunized with equivalent amounts of SEA or CFA alone displayed the smaller hepatic lesions in a Th2-dominant environment typically seen in the unimmunized BL/6 mice. Analysis of granuloma and MLN lymphocytes from the SEA/CFA-immunized mice revealed that the proportion of CD4 T cells was unchanged in comparison with the control BL/6 groups and remained significantly lower than that seen in the normally high-pathology CBA strain. These results suggest that the shift toward Th1-type cytokine production by a numerically stable population of CD4 T cells correlates with severe exacerbation of immunopathology in schistosomiasis.

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.

Exacerbated egg-induced immunopathology in murine Schistosoma mansoni infection is primarily mediated by IL-17 and restrained by IFN-γ.

In schistosomiasis, the severity of CD4+ T‐cell‐mediated hepatic granulomatous inflammation against parasite eggs varies considerably in humans and among mouse strains. In C57BL/6 mice, pronounced exacerbation of immunopathology induced by immunization with schistosome egg Ag in CFA (SEA/CFA) substantially recapitulates the natural high pathology seen in CBA mice; both are associated with a significant elevation of Th17‐ and Th1‐cell‐derived proinflammatory cytokines. We now investigated the relative contribution of the effector cytokines IL‐17 and IFN‐γ in pathology development of 7 wk‐infected, SEA/CFA‐immunized, IL‐17−/−, IFN‐γ−/−, and IL‐17/IFN‐γ−/− mice. In IL‐17−/− mice there was significant reduction of immunopathology despite increased levels of IFN‐γ, whereas in IFN‐γ−/− mice, markedly exacerbated immunopathology correlated with an increase in IL‐17. In IL‐17/IFN‐γ−/− mice, complete resistance to SEA/CFA‐induced disease exacerbation was associated with a reduction in IL‐23p19, IL‐1β, CXCL1 and iNOS, and with an increase in IL‐5, IL‐10 and Relmα. IL‐17 and IFN‐γ were derived from distinct CD4+ T cells in which production of each cytokine was suppressed by the other. Our results indicate that severe immunopathology in murine schistosomiasis is mainly driven by IL‐17 and regulated by IFN‐γ; however, in the absence of IL‐17, IFN‐γ is capable of exerting a limited, yet significant, pathogenic function.

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.