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Key Points Nlrp3−/− mice have enhanced early antihelminth immunity in the lung. Type 2 immunity and repair responses are dysregulated in Nlrp3−/− mice. NLRP3 plays an inflammasome-independent role during Nippostrongylus infection., Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R–dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis, suggesting a potential role for the inflammasome in the IL-1–mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3−/− mice with N. brasiliensis. Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3−/− mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3−/− mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis, and in contrast to Nlrp3−/− mice, antihelminth responses were unaffected in caspase-1/11–deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite–host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Objective. To examine if the protective effect of parasite infection on Experimental Autoimmune Encephalomyelitis (EAE) was due to interleukin-5, a cytokine produced by a Type-2 response that induces eosinophilia. We hypothesise that in parasite infections IL-5 also promotes expansion of antigen-specific T regulatory cells that control autoimmunity. Methods. Nippostrongylus brasiliensis larvae were used to infect Lewis rats prior to induction of EAE by myelin basic protein. Animals were sham-treated, or given blocking monoclonal antibodies to interleukin 4 or 5 or to deplete CD25+T cells. Reactivity of CD4+CD25+T regulatory cells from these animals was examined. Results. Parasite infected hosts had reduced severity and length of EAE. The beneficial effect of parasitic infection was abolished with an anti-IL-5 or an anti-CD25 monoclonal antibody, but not anti-IL-4 monoclonal antibody. Parasite infected animals with EAE developed antigen-specific CD4+CD25+T regulatory cells earlier than EAE controls and these expressed more Il5ra than controls. Treatment with IL-5 also reduced the severity of EAE and induced Il5ra expressing CD4+CD25+T regulatory cells. Interpretation. The results of this study suggested that IL-5 produced by the Type-2 inflammatory response to parasite infection promoted induction of autoantigen-specific CD25+Il5ra+T regulatory cells that reduced the severity of autoimmunity. Such a mechanism may explain the protective effect of parasite infection in patients with multiple sclerosis where eosinophilia is induced by IL-5, produced by the immune response to parasites.

Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP (L-GMP). Using an in vivo hematopoietic-specific gene-ablation model we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitors (MPP) were all significantly reduced in Klf5Δ/Δ mice, and knockdown of KLF5 in human CD34+ cells suppressed colony forming potential. ST-HSC, MPP and total numbers of committed progenitors were increased in the spleen of Klf5Δ/Δ mice, and reduced β1- and β2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis result from increased stem and progenitor mobilization. Klf5Δ/Δ mice show a significant reduction in the fraction of Gr1+Mac1+ cells (neutrophils) in peripheral blood and bone marrow, and increased frequency of eosinophils in the peripheral blood, bone marrow and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice post-GMP, promoting increased neutrophil output at the expense of eosinophil production. Refereed/Peer-reviewed

Expulsion of adult Nippostrongylus brasiliensis worms from the small intestine is profoundly impaired in signal transducer and activator of transcription (STAT)6-deficient mice. IL-5 transgenic (Tg) mice with constitutive eosinophilia show profound early resistance in the skin and/or later pre-lung phase of primary infections with N. brasiliensis. This study was designed to assess the importance of the eosinophil chemokine eotaxin and the STAT6/interleukin (IL)-4/IL-13 signalling pathway in early resistance to N. brasiliensis. Eosinophil recruitment into the skin following injection of N. brasiliensis larvae was reduced in STAT6- or eotaxin-deficient/IL-5 Tg double mutant mice. While ablation of eotaxin did not impair resistance in the pre-lung phase of N. brasiliensis infections in IL-5 Tg mice, elimination of STAT6 caused a modest reduction in resistance in both primary and secondary infections on this genetic background. STAT6(-/-)-, IL-13(-/-)- and IL-4Ralpha(-/-)-deficient single mutant and IL-13(-/-)/IL-4Ralpha(-/-) double mutant mice were more susceptible than WT mice during the pre-lung phase of secondary N. brasiliensis infections. In contrast, primary or secondary resistance were unaffected at either the pre-lung or gut stages of infection in eotaxin(-/-) single mutant mice. STAT6(-/-) and eotaxin(-/-) mice with or without the IL-5 transgene, were no more susceptible than WT or IL-5 Tg mice to protracted primary infections with Heligmosomoides bakeri, a parasitic nematode that is restricted to the gut. Our data suggest that parasitic nematodes that transit through the skin and lungs en route to the gut may be susceptible to early (pre-lung) innate and adaptive immune mechanisms that are dependent on the STAT6/IL-4/IL-13 signalling pathway, and this may be important for the development of effective therapies and vaccines.

Humans vary markedly in their propensity to develop asthma, despite often being exposed to similar environmental stimuli. Similarly, mouse strains vary in susceptibility to airways pathology in experimental asthma. Sensitization and aerosol challenge with ovalbumin (OVA) induces eosinophil accumulation, mucus production and airways obstruction in BALB/c and C57BL/6 mice. In contrast, CBA/Ca mice show relatively little pathology. Allergen-induced production of IL-4, IL-5, IL-10 and IFN-γ was detected in all three strains, with BALB/c mice generating the highest levels of IL-4, IL-5 and IL-10. Microarray analysis was used to identify genes differentially regulated in lung tissue after OVA challenge. Differentially regulated genes in the lungs of the asthma-susceptible C57BL/6 and BALB/c strains numbered 242 and 145, respectively, whereas only 42 genes were differentially expressed in the resistant CBA/Ca strain. In C57BL/6 mice, transcripts were enriched for adhesion molecules and this was associated with high levels of eosinophil recruitment. Differentially regulated genes in the lungs of only the asthma-susceptible strains numbered 64 and several of these have not previously been associated with asthma. Many of the genes differentially regulated in the susceptible strains were enzymes involved in inflammation. Using network analysis, mRNA for the anti-apoptotic protein survivin was found to be up-regulated in the lungs following allergen challenge. Survivin mRNA and protein were also expressed at high levels in eosinophils recovered by bronchoalveolar lavage from BALB/c and C57BL/6 mice. We propose that rapid apoptosis of lung eosinophils due to low expression of survivin contributes to the limitation of pathology in CBA/Ca mice. Refereed/Peer-reviewed

SUMMARYNippostrongylus brasiliensislarvae are particularly susceptible to immunological attack during the pre-lung stage of primary and secondary infections in mice. Whilst most of the common laboratory strains of mice are permissive hosts for the parasite, in this study we report for the first time, the strong resistance of naïve FVB/N mice toN. brasiliensis. Damage to larvae is evident within the first 24 h of infection and this may be critical to later larval development and reproductive success. Inflammatory responses in the skin, and larval escape from this tissue were comparable in susceptible CBA/Ca and resistant FVB/N mice, with most larvae exiting within 4 h of a primary infection. Lung larval burdens were also similar between strains, but larvae recovered from FVB/N mice were smaller and less motile. In FVB/N mice, larval colonization of the gut was impaired and worms produced very few eggs. However FVB/N mice did not show enhanced resistance toHeligmosomoides bakeri(also known asHeligmosomoides polygyrus), a nematode largely restricted to the gut. Damage done in the pre-lung or lung stages of infection withN. brasiliensisis likely to contribute to ongoing developmental and functional abnormalities, which are profoundly evident in the gut phase of infection.

Complement may be important for immunity to infection with parasitic helminths, by promoting the recruitment of leukocytes to infected tissues and by modulating the function of cytotoxic effector leukocytes. However, the importance of complement in vivo during helminth infection is poorly understood. In this study, mice lacking classical (C1q-deficient), alternative (factor B-deficient) or all pathways of complement activation (C3-deficient) were used to assess the role of complement in immunity to the nematode Nippostrongylus brasiliensis. Double-mutant complement-deficient/IL-5 transgenic (Tg) mice were used to determine if complement is required for the strong eosinophil-dependent resistance to this parasite. Complement activation on larvae (C3 deposition), extracellular eosinophil peroxidase activity, larval aggregation and eosinophil recruitment to the skin 30 min post-injection (p.i.) of larvae were reduced in factor B-deficient mice. Inhibition of the C5a receptor with the antagonist PMX53 impaired eosinophil and neutrophil recruitment to the skin. C3 deposition on larvae was minimal by 150 min p.i. and at this time cell adherence, larval aggregation, eosinophil recruitment and degranulation were complement-independent. Factor B and C3 deficiency were associated with higher lung larval burdens in primary infections. Complement-deficient/IL-5 Tg mice were highly resistant to N. brasiliensis, suggesting that eosinophils can limit infection in a complement-independent manner. Potent secondary immunity was similarly complement-independent. In conclusion, although the alternative pathway is important for parasite recognition and leukocyte recruitment early in N. brasiliensis infections, the parasite soon becomes resistant to complement and other factors can compensate to promote eosinophil-dependent immunity.

Extracellular cyclophilins have been well described as chemotactic factors for various leukocyte subsets. This chemotactic capacity is dependent upon interaction of cyclophilins with the cell surface signaling receptor CD147. Elevated levels of extracellular cyclophilins have been documented in several inflammatory diseases. We propose that extracellular cyclophilins, via interaction with CD147, may contribute to the recruitment of leukocytes from the periphery into tissues during inflammatory responses. In this study, we examined whether extracellular cyclophilin-CD147 interactions might influence leukocyte recruitment in the inflammatory disease allergic asthma. Using a mouse model of asthmatic inflammation, we show that 1) extracellular cyclophilins are elevated in the airways of asthmatic mice; 2) mouse eosinophils and CD4+ T cells express CD147, which is up-regulated on CD4+ T cells upon activation; 3) cyclophilins induce CD147-dependent chemotaxis of activated CD4+ T cells in vitro; 4) in vivo treatment with anti-CD147 mAb significantly reduces (by up to 50%) the accumulation of eosinophils and effector/memory CD4+ T lymphocytes, as well as Ag-specific Th2 cytokine secretion, in lung tissues; and 5) anti-CD147 treatment significantly reduces airway epithelial mucin production and bronchial hyperreactivity to methacholine challenge. These findings provide a novel mechanism whereby asthmatic lung inflammation may be reduced by targeting cyclophilin-CD147 interactions.

Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCE This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.

Complement activation and C3 deposition on the surface of parasitic helminths may be important for recruitment of leukocytes and for damage to the target organism via cell-mediated mechanisms. Inhibition of complement activation would therefore be advantageous to parasites, minimizing damage and enhancing migration through tissues. The aim of this study was to determine ex vivo if complement activation by, and leukocyte adherence to, the nematodeNippostrongylus brasiliensischange as the parasite matures and migrates through the murine host. Pathways of activation of complement and the mechanism of adherence of leukocytes were also defined using sera from mice genetically deficient in either C1q, factor B, C1q and factor B, C3, or C4. Substantive deposition of C3 and adherence of eosinophil-rich leukocytes were seen with infective-stage (L3) but not with lung-stage (L4) larvae. Adult intestinal worms had low to intermediate levels of both C3 and leukocyte binding. For L3 and adult worms, complement deposition was principally dependent on the alternative pathway. For lung-stage larvae, the small amount of C3 detected was dependent to similar degrees on both the lectin and alternative pathways. The classical pathway was not involved for any of the life stages of the parasite. These results suggest that in primary infections, the infective stage ofN. brasiliensisis vulnerable to complement-dependent attack by leukocytes. However, within the first 24 h of infection,N. brasiliensisacquires the ability to largely avoid complement-dependent immune responses.

Methods used to quantify complement deposition and cell adherence to parasitic helminths usually involve subjective visual comparisons of immunofluorescence or time-consuming manual counting of bound cells. Such targets are relatively large and, generally, few individual organisms can be analysed. More objective and efficient radiometric assays are available, but these also have considerable disadvantages. We have developed an improved immunofluorescence-based method for quantitation of complement deposition on viable third-stage larvae of the nematode Nippostrongylus brasiliensis (L3). A similar strategy was also applied to measuring leukocyte adherence to the parasite. Fluorescein isothiocyanate (FITC)-conjugated antibodies were used to detect complement on serum-treated larvae. The adherence of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled mouse leukocytes to larvae was investigated using the same basic approach. Images of fluorescent larvae or fluorescent cells attached to larvae were generated with a Bio-Rad Molecular Imager FX and fluorescence intensity was quantified. Hundreds of larvae can be analysed simultaneously in multiple samples, and these strategies allow rapid and sensitive quantitation that is directly proportional to the amount of protein or the number of leukocytes added to cultures. These techniques may also be applicable to other large objects, organisms or biological surfaces.

Eosinophil degranulation is thought to play a pivotal role in the pathogenesis of allergic disorders. Although mouse models of allergic disorders have been used extensively to identify the contribution of eosinophils to disease, ultrastructural evidence of active granule disassembly has not been reported. In this investigation, we characterized the degree of eosinophil activation in the bone marrow, blood, lung tissue, and airways lumen [bronchoalveolar lavage fluid (BALF)] of ovalbumin-sensitized and aero-challenged wild-type and interleukin-5 transgenic mice. Degranulation was most prominent in and primarily compartmentalized to the airways lumen. Eosinophils released granule proteins by the process of piecemeal degranulation (PMD). Accordingly, recruitment and activation of eosinophils in the lung correlated with the detection of cell-free eosinophil peroxidase in BALF and with the induction of airways hyper-reactivity. As in previous studies with human eosinophils, degranulation of isolated mouse cells did not occur until after adherence to extracellular matrix. However, higher concentrations of exogenous stimuli appear to be required to trigger adherence and degranulation (piecemeal) of mouse eosinophils when compared with values reported for studies of human eosinophils. Thus, mouse eosinophils undergo PMD during allergic inflammation, and in turn, this process may contribute to pathogenesis. However, the degranulation process in the allergic lung of mice is primarily compartmentalized to the airway lumen. Understanding the mechanism of eosinophil degranulation in the airway lumen may provide important insights into how this process occurs in human respiratory diseases.

Background Eotaxin-2 is a member of the eotaxin subfamily of CC chemokines that display eosinophil-specific, chemotactic properties and has been associated with allergic disorders. However, the contribution of eotaxin-2 to the development of defined pathogenic features of allergic disease remains to be defined. Objective We sought to determine whether eotaxin-2 was a cofactor with IL-5 for the regulation of pulmonary eosinophilia and to identify the combined role of these molecules in the induction of phenotypic characteristics of allergic lung disease. Methods We instilled recombinant eotaxin-2 into the airways of wild-type mice that had been treated systemically with IL-5 or into IL-5-transgenic mice and characterized pulmonary eosinophil numbers, IL-13 production, and airway hyperreactivity (AHR) to methacholine. Mice deficient in the IL-4 receptor α-chain, IL-13, and signal transducers and activators of transcription 6 or mice treated with anti-CCR3 monoclonal antibody were also used. Results Eotaxin-2 and IL-5 cooperatively promoted eosinophil accumulation, IL-13 production, and AHR to methacholine. Neither eotaxin-2 nor IL-5 alone induced these features of allergic disease. IL-13 production was critically dependent on eotaxin-2- and IL-5-regulated eosinophilia, which predisposed to the development of AHR. AHR was dependent on IL-13 and signaling through the IL-4R α-chain and signal transducers and activators of transcription 6 pathways and the presence of eosinophils in the lung. Conclusion These investigations demonstrate important cooperativity between eotaxin-2, IL-5, and IL-13 signaling systems and eosinophils for the development of hallmark features of allergic disease of the lung.

Eosinophils are prevalent in the female reproductive tract, where they may contribute to regulation of development and maintenance of epithelial integrity. The present study examined the effects of constitutive interleukin-5 (IL-5) expression and overabundance of eosinophils on the development and function of the mammary gland, uterus, and ovary in mice. Eosinophils were up to 13-fold and 4-fold more abundant in the uterus and mammary gland, respectively, in female IL-5 transgenic (IL-5Tg) mice than in wild-type (Wt) animals. Eosinophils were present in large numbers in regressing corpora lutea in IL-5Tg mice but not in ovaries from Wt mice. Postpubertal mammary gland development was retarded in IL-5Tg mice, with impaired terminal end bud formation and an altered pattern of epithelial cell proliferation across the mammary fat pad coincident with disrupted ductal branching and extension. By 10 wk of age, the ductal tree was complete in both genotypes. Onset of first estrus was also delayed in IL-5Tg mice, but once IL-5Tg mice reached puberty, serum estrogen content across the cycle and estrous cycle duration were normal. The histology of uterine tissue and epithelial cell turnover were unchanged. Capacity to mate and achieve pregnancy was not affected by maternal IL-5 transgene expression, although at Day 18 of gestation, a modest decrease in the fetal:placental weight ratio was observed. Furthermore, parturition and ability to lactate and nurture postnatal pup development were not compromised. These data demonstrate an effect of IL-5 overexpression on ductal morphogenesis during postpubertal mammary gland development that is consistent with a direct regulatory role for eosinophils in these events, but these data also show that eosinophil excess does not have long-term consequences for adult reproductive function.

Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease.

Eosinophils have long been thought to be effectors of immunity to helminths but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are functionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminths present a number of apparent paradoxes. Eosinophil accumulation in tissues adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasiliensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results also suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

Summary A relatively small number of laboratories in Australia and New Zealand have consistently published on murine models of nematode immunology, and the parasite species principally used are Heligmosomoides bakeri (previously Heligmosomoides polygyrus), Strongyloides ratti, Nippostrongylus brasiliensis and Toxocara canis. These research groups have made significant contributions to both fundamental immunology and more specialized issues in host–parasite relationships. Topics addressed include immune regulation, including the expression and control of Type 2 cytokines and the responses induced, innate and adaptive host-protective mechanisms, antigen expression and immune evasion strategies utilized by parasitic helminths. This review addresses the last 30 years of research and identifies areas in which major progress can be made, given appropriate resources.

Leucocytes are essential in healing wounds and are predominantly involved in the inflammatory and granulation stages of wound repair. Eosinophils are granulocytic leucocytes and are specifically regulated by interleukin-5 (IL-5), a cytokine produced by T helper 2 (Th2) cells. To characterize more clearly the role of the IL-5 and eosinophils in the wound healing process, IL-5-overexpressing and IL-5-deficient mice were used as models of eosinophilia and eosinophil depletion, respectively. Our results reveal a significantly altered inflammatory response between IL-5-overexpressing and IL-5 knockout mice post-wounding. Healing was significantly delayed in IL-5-overexpressing mice with wounds gaping wider and exhibiting impaired re-epithelialization. A delay in collagen deposition was observed suggesting a direct effect on matrix synthesis. A significant increase in inflammatory cell infiltration, particularly eosinophils and CD4+ cells, one of the main cell types which secrete IL-5, was observed in IL-5-overexpressing mice wounds suggesting that one of the main roles of IL-5 in wound repair may be to promote the infiltration of eosinophils into healing wounds. Healing is delayed in IL-5-overexpressing mice and this corresponds to significantly increased levels of eosinophils and CD4+ cells within the wound site that may contribute to and exacerbate the inflammatory response, resulting in detrimental wound repair. Refereed/Peer-reviewed

Survival of parasitic helminths within a host requires immune evasion and excretorylsecretory (ES) proteins may contribute to this process. Eosinophils are important effector cells in immunity of mice to the nematode Nippostrongylus brasiliensis and eosinophilic interleukin-5 transgenic (IL-5 Tg) mice are highly resistant to the earliest stages of primary infections. In contrast, Toxocara canis is largely resistant to eosinophils, with viable larvae encysted in tissues often surrounded by these and other leucocytes. The aim of this study was to investigate whether T. canis ES (TES) proteins inhibit eosinophil-dependent resistance to N. brasiliensis. Mouse serum pre-treated with TES had reduced capacity to mediate the adherence of leucocytes to N. brasiliensis infective-stage larvae (L3) and this correlated with reduced complement C3 deposition on the parasite. TES did not inhibit eosinophil survival or eotaxin-dependent eosinophil migration in vitro. Cellular inflammation and eosinophil degranulation in the skin in response to injection of L3 was also not impaired by TES. However, when TES was included with L3 in an inoculum given to IL-5 Tg mice, a greatly increased number of parasites migrated to the lung. This suggests that the early eosinophil-dependent resistance in these mice was suppressed, by mechanisms yet to be determined. Refereed/Peer-reviewed

The accumulation of eosinophils is a common feature of allergic airway inflammation and correlates with disease severity. In an ovalbumin (OVA)-induced murine model of allergic lung disease, CBA/Ca mice develop much lower levels of lung eosinophilia, lung oedema, mucus hypersecretion and airways obstruction than BALB/c and C57BL/6 strains. In this study these strains have been examined to identify mechanisms that control the recruitment and survival of eosinophils in the allergic lung. Following immunization with OVA, CBA/Ca mice developed a robust systemic allergic response, with high levels of total and OVA-specific IgE and increases in peripheral blood eosinophils. Lung eotaxin-1 levels and expression of CD18 on eosinophils recovered by bronchoalveolar lavage (BAL) were least pronounced in CBA/Ca mice, whereas mRNA for L-selectin was highest in eosinophils from C57BL/6 mice. Apoptosis of BAL eosinophils ex vivo was most pronounced in the CBA/Ca strain. BALB/c mice expressed the highest levels of the eosinophil growth and survival factor interleukin (IL)-5 in the lungs and BAL eosinophils from these animals expressed more of the anti-apoptotic proteins Bcl-x L and Bcl-2 than cells from the other strains. A combination of lower levels of recruitment and rapid apoptosis may therefore limit the accumulation of eosinophils and pathology in the lungs of CBA/Ca mice. In addition, although the level of pathology that developed in C57BL/6 and BALB/c mice was similar, some of the underlying mechanisms are likely to differ. Refereed/Peer-reviewed

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.

Although exposed to similar allergic and environmental stimuli, not all humans develop asthma. Similarly, mouse strains vary in the degree of pathophysiology seen following induction of experimental asthma. Three mouse strains (CBA/Ca, BALB/c, and C57BL/6) were used to determine if the extent and duration of inflammation influenced the degree of lung tissue damage in an OVA-induced allergic asthma model. Airways obstruction, leukocyte infiltration, edema, eosinophil accumulation, and degranulation were less severe in wild-type (wt) CBA/Ca mice than wt BALB/c and C57BL/6 mice. F1 hybrids of CBA/Ca mice crossed with BALB/c or C57BL/6 mice had bronchoalveolar lavage leukocyte (BAL) and cell-free protein profiles similar to those of the respective disease-susceptible parental strain. IL-5 transgene expression on each of the three genetic backgrounds accentuated the difference between CBA/Ca and the other two strains. Importantly, even when overexpressing IL-5, CBA/Ca mice did not develop substantial airways obstruction. Eosinophils recovered from the airways of allergic wt and IL-5 transgenic (Tg) CBA/Ca mice entered apoptosis at a faster rate than eosinophils from the other parental strains and F1 hybrids. In contrast, eosinophils harvested from the peritoneal cavities of untreated CBA/Ca IL-5 Tg mice had a relatively low rate of apoptosis in vitro. The CBA/Ca mouse strain is therefore relatively resistant to experimental asthma, and this may be a consequence of a propensity for apoptosis of eosinophils recruited into the allergic lung. Restricting survival of a key effector cell may thus limit pathogenesis in this experimental model and in humans.

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11−/− and ΔdblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11−/− BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/CCL11−/− and ΔdblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance.

Eosinophils are an important feature of immune responses to infections with many of the tissue-invasive helminth parasites. The cytokine IL-5 and a high-affinity double GATA-binding site within the GATA-1 promoter are critical for eosinophilopoiesis. In this study, we believe we demonstrate for the first time that defects in eosinophilopoiesis are associated with impaired resistance to Nippostrongylus brasiliensis. Primary and secondary infections were established in wildtype (WT), IL-5(-/-) and DeltadblGATA mice. Resistance to secondary infections was impaired in IL-5(-/-) and DeltadblGATA mice, with significantly more larvae able to reach the lungs 2 days p.i. Pulmonary inflammation was minimal in all strains in the first 2 days of both primary and secondary infections, suggesting that eosinophil-dependent resistance occurred before larvae reached this site. Intestinal worm burdens and/or parasite egg production in primary infections were greater in animals with defective eosinophilopoiesis. While larvae did reach the gut by day 3 of secondary infections of WT and IL-5(-/-) mice, worms were expelled by day 7, even in the complete absence of eosinophils in tissues of the small intestine. This and our previous studies indicate that N. brasiliensis are likely to be exquisitely sensitive to attack by eosinophils soon after entry into the skin. Eosinophils in the gut may make a modest contribution to resistance on first exposure to the parasite, but are not required for expulsion in either primary or secondary infections. In order to mount an effective immune response it may be vital for the host to identify and attack the parasite before it implements immune evasion strategies and migrates to other anatomical sites. These observations may be of particular significance for the development of successful vaccines against hookworms and other nematodes.

Eosinophilic inflammation is a common feature of numerous eosinophil-associated gastrointestinal (EGID) diseases. Central to eosinophil migration into the gastrointestinal tract are the integrin-mediated interactions with adhesion molecules. Although the mechanisms regulating eosinophil homing into the small intestine have begun to be elucidated, the adhesion pathways responsible for eosinophil trafficking into the large intestine are unknown. We investigated the role of adhesion pathways in eosinophil recruitment into the large intestine during homeostasis and disease. First, using a hapten-induced colonic injury model, we demonstrate that in contrast to the small intestine, eosinophil recruitment into the colon is regulated by a β7-integrin addressin cell adhesion molecule-1-independent pathway. Characterization of integrin expression on colonic eosinophils by flow cytometry analysis revealed that colonic CC chemokine receptor 3+ eosinophils express the intercellular adhesion molecule-1 (ICAM-1) counter-receptor integrins αL, αM, and β2. Using ICAM-1-deficient mice and anti-ICAM-1 neutralizing antibodies, we show that hapten-induced colonic eosinophilic inflammation is critically dependent on ICAM-1. These studies demonstrate that β2-integrin/ICAM-1-dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.

Eosinophils are prevalent in the female reproductive tract, where they may contribute to regulation of development and maintenance of epithelial integrity. The present study examined the effects of constitutive interleukin-5 (IL-5) expression and overabundance of eosinophils on the development and function of the mammary gland, uterus, and ovary in mice. Eosinophils were up to 13-fold and 4-fold more abundant in the uterus and mammary gland, respectively, in female IL-5 transgenic (IL-5Tg) mice than in wild-type (Wt) animals. Eosinophils were present in large numbers in regressing corpora lutea in IL-5Tg mice but not in ovaries from Wt mice. Postpubertal mammary gland development was retarded in IL-5Tg mice, with impaired terminal end bud formation and an altered pattern of epithelial cell proliferation across the mammary fat pad coincident with disrupted ductal branching and extension. By 10 wk of age, the ductal tree was complete in both genotypes. Onset of first estrus was also delayed in IL-5Tg mice, but once IL-5Tg mice reached puberty, serum estrogen content across the cycle and estrous cycle duration were normal. The histology of uterine tissue and epithelial cell turnover were unchanged. Capacity to mate and achieve pregnancy was not affected by maternal IL-5 transgene expression, although at Day 18 of gestation, a modest decrease in the fetal:placental weight ratio was observed. Furthermore, parturition and ability to lactate and nurture postnatal pup development were not compromised. These data demonstrate an effect of IL-5 overexpression on ductal morphogenesis during postpubertal mammary gland development that is consistent with a direct regulatory role for eosinophils in these events, but these data also show that eosinophil excess does not have long-term consequences for adult reproductive function.

What represents a protective or beneficial immune response in one scenario, may contribute to the pathogenesis of disease in another. This review explores the plasticity of immune responses and the delicate balance between health and disease, using examples from immunoparasitology, allergic lung disease and reproductive biology. Cytokines secreted by lymphocytes and other leukocytes are central to this balance because they regulate both inflammation and adaptive immunity. The type and quantity of cytokines, the timing and location of cytokine release, and coordinated expression with other signals can all contribute in determining the nature of immune responses and, therefore, of disease outcomes. Of necessity, leukocytes control and eliminate infectious agents by interacting with other cells. However, leukocytes also communicate with other cells to maintain homeostasis in healthy organisms. Tissue development, repair, remodelling and immunopathology can be viewed as parts of a continuum and leukocytes are major contributors to all of these processes. The factors, which influence the extent to which an infection will result in host pathology, are multifarious, but include as yet poorly determined elements within the genetic background of the host. Nowhere is this more obvious than in animals chronically infected with parasites. There are parallels between parasite infections and pregnancy, since to survive and develop, the conceptus also must avoid immunological rejection. Therapeutic intervention through manipulation of cytokine profiles may be feasible, but is fraught with risk and should not be undertaken without careful analysis of the possible consequences in a range of genetic backgrounds and with consideration of the diversity of infectious agents which might be encountered.

Eosinophils are primarily thought of as terminal effectors of allergic responses and of parasite elimination. However, limited studies suggest a more discrete immunomodulatory role for this leukocyte during these inflammatory responses. In this investigation, we highlight the potential of eosinophils to act as APCs and thus modulators of allergic responses by influencing Th2 cell function. In response to Ag provocation of the allergic lung, eosinophils rapidly trafficked to sites of Ag deposition (airways lumen) and presentation (lung-associated lymph nodes and T cell-rich paracortical zones). Eosinophils from the allergic lung expressed class II MHC peptides, T cell costimulatory molecules (CD80 and CD86), and rapidly internalized and processed Ag that was sampled from within the airway lumen. Ag-loaded eosinophils promoted the production of IL-4, IL-5, and IL-13 in cocultures with in vitro-polarized Th2 cells and induced IL-5 production in a dose-dependent manner from Ag-specific CD4+ T cells isolated from allergic mice. In addition, Ag-loaded eosinophils primed for Th2 cell-driven allergic disease of the lung when transferred to naive mice. Thus, eosinophils have the potential to not only activate Th2 cells to release disease-modulating cytokines but also to assist in priming the immune system for allergic responses. This investigation highlights the potential of eosinophils to not only act as terminal effector cells but also to actively modulate allergic inflammation by amplifying Th2 cell responses.

Interleukin-5 (IL-5) transgenic mice are highly resistant to primary infections with the intestinal nematode Nippostrongylus brasiliensis ; few parasites are found in the intestines of infected animals, and egg production is minimal. While adult worms may be damaged in the intestine, larval migration, development, and viability may also be impaired in other tissues. This study addresses the migration of N. brasiliensis larvae through the skin and lungs and associated cellular responses in primary infections of IL-5 transgenic mice. Although some larvae may have been trapped and killed in the lungs of IL-5 transgenic mice, most apparently failed to reach this site. Two or more hours after infection of IL-5 transgenic mice, eosinophils were a major component of the cellular infiltrate at the subcutaneous site of injection, and localized eosinophil degranulation was extensive. Seventy-five to ninety-five percent of the larvae injected into subcutaneous air pouches in IL-5 transgenic mice were retained there for at least 24 h. In contrast, in nontransgenic mice, less than 20% of larvae could be recovered from the skin 2 or more h postinjection, and eosinophil activity was modest at all times. The data strongly suggest that eosinophils can restrict the movement of N. brasiliensis larvae in the first few hours of a primary infection and that this has profound effects on later stages of parasite development. Preexisting eosinophilia, due either to allergy or to infection with tissue-invasive helminth species, may therefore confer some degree of immediate and nonspecific resistance in primary infections with parasitic worms.

In this study, interleukin-5 (IL-5) transgenic mice with lifelong eosinophilia were assessed for resistance to primary infections with two tissue-invading nematodes, Nippostrongylus brasiliensis and Toxocara canis . Relative to nontransgenic littermates, three lines of IL-5 transgenic mice with varying degrees of eosinophilia all displayed enhanced resistance to N. brasiliensis . Although the timing of final worm expulsion was similar in transgenic and nontransgenic hosts, intestinal worms in transgenic mice were fewer in number throughout infection, failed to increase in size over the course of the infection, and were much less fecund. In contrast, T. canis larvae were recovered in similar numbers from tissues of transgenic mice with “low” or “high” eosinophilia and from nontransgenic mice. These results and other data suggest that eosinophils can contribute to host resistance to some parasite species. Parasite transit time through the host may correlate with relative sensitivity to eosinophils.

Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths. This study examined the possible influence of constituitive eosinophilia on the clearance of Schistosoma mansoni infections in mice. Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure, and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli. IL-5 transgenics and non-transgenic littermates were immunized once or four (hyperimmunization) times with irradiated cercariae of S. mansoni. Animals were challenged percutaneously with unirradiated cercariae one month after their last exposure to irradiated parasites. One month after challenge transgenic animals, whether unimmunized, vaccinated or hypervaccinated, carried significantly more liver-stage parasites than non-transgenic animals. These results suggest that although eosinophils from IL-5 transgenic mice are functional for a number of key parameters, large numbers of eosinophils and/or high levels of IL-5 may in some way impair clearance of S. mansoni. A re-evaluation of the roles of eosinophils and IL-5 in infections with this and other parasites may therefore be warranted.