Robin J. Flynn

Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity

Innate immunity provides the first line of defence against invading pathogens and provides important cues for the development of adaptive immunity. Type-2 immunity—responsible for protective immune responses to helminth parasites and the underlying cause of the pathogenesis of allergic asthma—consists of responses dominated by the cardinal type-2 cytokines interleukin (IL)4, IL5 and IL13 (ref. 5). T cells are an important source of these cytokines in adaptive immune responses, but the innate cell sources remain to be comprehensively determined. Here, through the use of novel Il13-eGFP reporter mice, we present the identification and functional characterization of a new innate type-2 immune effector leukocyte that we have named the nuocyte. Nuocytes expand in vivo in response to the type-2-inducing cytokines IL25 and IL33, and represent the predominant early source of IL13 during helminth infection with Nippostrongylus brasiliensis. In the combined absence of IL25 and IL33 signalling, nuocytes fail to expand, resulting in a severe defect in worm expulsion that is rescued by the adoptive transfer of in vitro cultured wild-type, but not IL13-deficient, nuocytes. Thus, nuocytes represent a critically important innate effector cell in type-2 immunity.

IL-33 is more potent than IL-25 in provoking IL-13–producing nuocytes (type 2 innate lymphoid cells) and airway contraction

BACKGROUND IL-25 and IL-33 belong to distinct cytokine families, but experimental mouse studies suggest their immunologic functions in type 2 immunity are almost entirely overlapping. However, only polymorphisms in the IL-33 pathway (IL1RL1 and IL33) have been significantly associated with asthma in large-cohort genome-wide association studies. OBJECTIVE We sought to identify distinct pathways for IL-25 and IL-33 in the lung that might provide insight into their roles in asthma pathogenesis and potential for therapeutic intervention. METHODS IL-25 receptor-deficient (Il17rb(-/-)), IL-33 receptor-deficient (ST2, Il1rl1(-/-)), and double-deficient (Il17rb(-/-)Il1rl1(-/-)) mice were analyzed in models of allergic asthma. Microarrays, an ex vivo lung slice airway contraction model, and Il13(+/eGFP) mice were then used to identify specific effects of IL-25 and IL-33 administration. RESULTS Comparison of IL-25 and IL-33 pathway-deficient mice demonstrates that IL-33 signaling plays a more important in vivo role in airways hyperreactivity than IL-25. Furthermore, methacholine-induced airway contraction ex vivo increases after treatment with IL-33 but not IL-25. This is dependent on expression of the IL-33 receptor and type 2 cytokines. Confocal studies with Il13(+/eGFP) mice show that IL-33 more potently induces expansion of IL-13-producing type 2 innate lymphoid cells, correlating with airway contraction. This predominance of IL-33 activity is enforced in vivo because IL-33 is more rapidly expressed and released in comparison with IL-25. CONCLUSION Our data demonstrate that IL-33 plays a critical role in the rapid induction of airway contraction by stimulating the prompt expansion of IL-13-producing type 2 innate lymphoid cells, whereas IL-25-induced responses are slower and less potent.

IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis

Significance Abnormal damage and scarring of tissue (fibrosis) in the lungs can lead to pulmonary fibrosis. Patients that develop the various forms of pulmonary fibrosis are difficult to treat and have a high level of mortality. In this study we have used mouse models to address the role of the cytokine interleukin (IL)-25 and type 2 innate lymphoid cells (ILC2) in pulmonary fibrosis. In animal models we show a role for IL-25 and ILC2 in the generation of pulmonary fibrosis. Furthermore, we have identified elevated levels of IL-25 and ILC2s in the lungs of patients with idiopathic pulmonary fibrosis. This study provides insights on the factors and cells that may initiate pulmonary fibrosis in humans and have therapeutic potential. Disease conditions associated with pulmonary fibrosis are progressive and have a poor long-term prognosis with irreversible changes in airway architecture leading to marked morbidity and mortalities. Using murine models we demonstrate a role for interleukin (IL)-25 in the generation of pulmonary fibrosis. Mechanistically, we identify IL-13 release from type 2 innate lymphoid cells (ILC2) as sufficient to drive collagen deposition in the lungs of challenged mice and suggest this as a potential mechanism through which IL-25 is acting. Additionally, we demonstrate that in human idiopathic pulmonary fibrosis there is increased pulmonary expression of IL-25 and also observe a population ILC2 in the lungs of idiopathic pulmonary fibrosis patients. Collectively, we present an innate mechanism for the generation of pulmonary fibrosis, via IL-25 and ILC2, that occurs independently of T-cell–mediated antigen-specific immune responses. These results suggest the potential of therapeutically targeting IL-25 and ILC2 for the treatment of human fibrotic diseases.

Experimental Fasciola hepatica Infection Alters Responses to Tests Used for Diagnosis of Bovine Tuberculosis

ABSTRACT Fasciola hepatica is a prevalent helminth parasite of livestock. Infection results in polarization of the hosts immune response and generation of type 2 helper (Th2) immune responses, which are known to be inhibitory to Th1 responses. Bovine tuberculosis (BTB) is a bacterial disease of economic and zoonotic importance. Control polices for this disease rely on extensive annual testing and a test-and-slaughter policy. The correct diagnosis of BTB relies on cell-mediated immune responses. We established a model of coinfection of F. hepatica and Mycobacterium bovis BCG to examine the impact of helminth infection on correct diagnosis. We found the predictive capacity of tests to be compromised in coinfected animals and that F. hepatica infection altered macrophage function. Interleukin-4 and gamma interferon expression in whole-blood lymphocytes restimulated in vitro with M. bovis antigen was also altered in coinfected animals. These results raise the question of whether F. hepatica infection can affect the predictive capacity of tests for the diagnosis of BTB and possibly also influence susceptibility to BTB and other bacterial diseases. Further studies on the interplay between helminth infection and BTB are warranted.

Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1).

The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, Montanide ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p<or=0.05. All vaccinated animals showed a sharp rise in total IgG levels to rFhCL1 post-vaccination which was maintained over the course of the 13-week challenge infection and was significantly higher than levels reached in the control group. Arginase levels in the macrophages of vaccinated cattle were significantly lower than those of the control cattle, indicating that the parasite-induced alternative-activation of the macrophages was altered by vaccination. The data demonstrate the potential for recombinant FhCL1 vaccine in controlling fasciolosis in cattle under field conditions.

The roles of IL-10 and TGF-β in controlling IL-4 and IFN-γ production during experimental Fasciola hepatica infection

Hosts infected with Fasciola hepatica experience immunosuppression during the acute and chronic phases of the disease. This immunosuppression may allow parasite survival in the face of an ongoing immune response. In bovine hosts early IL-4 and continued IgG1 production is one of the few remaining features of the characteristic type 0/2 helper (Th0/2) response present in the chronic stage of disease. Here we demonstrate elevated levels of parasite-specific, in vitro peripheral blood mononuclear cell (PBMC)-derived transforming growth factor (TGF)-beta1 from the early phases of infection and increasing levels of IL-10 as the infection becomes chronic. In vitro neutralisation of these cytokines during culture of PBMCs from experimentally-infected cattle increased IL-4 and IFN-gamma production in response to parasite-specific and non-specific stimulation. At 4 weeks p.i. neutralisation of TGF-beta results in an increase in parasite driven IL-4, while also having a greater role, compared with IL-10, in influencing specific and non-specific IFN-gamma. At 12 weeks p.i. ex vivo parasite driven IL-4 was not restored by inhibiting either IL-10 or TGF-beta. However IL-10 influenced both parasite-specific and non-specific IFN-gamma production at this time. This highlights the roles of IL-10 and TGF-beta in fasciolosis, however the cellular sources of these have yet to be defined. This suggests that suppression of IFN-gamma production by parasite molecules occurs during infection and it is possible that the suppression of IFN-gamma production may mediate parasite survival in this disease.

Reciprocal expression of IL-25 and IL-17A is important for allergic airways hyperreactivity

Background Interleukin (IL)‐25 (IL‐17E) is a potent inducer of the type‐2 immune effector response. Previously we have demonstrated that a neutralizing anti‐IL‐25 antibody, given during the establishment of ovalbumin‐specific lung allergy, abrogates airways hyperreactivity.

Possible Role for Toll-Like Receptors in Interaction of Fasciola hepatica Excretory/Secretory Products with Bovine Macrophages

ABSTRACT Alternative activation of macrophages (Mφ) during helminth infection is a characteristic feature of the host immune response. Alternatively activated macrophages (AAMφ) are distinguished from others by high arginase 1 (Arg-1) activity, low nitric oxide (NO), and high interleukin 10 (IL-10) production. In murine models, these cells have been shown to possess anti-inflammatory properties. They have also been implicated in exacerbating a subsequent infection with a secondary pathogen. In this study we used cattle experimentally infected with Fasciola hepatica to monitor the kinetics of IL-4 and IL-10 over the course of infection. Using naïve Mφ in vitro, we examined the effects of exposure to F. hepatica excretory/secretory products (FhepES) alone or in combination with IL-4. Our results suggest that FhepES may work in combination with IL-4 to produce AAMφ. The effects of FhepES on the subsequent responses to lipopolysaccharide (LPS) and purified protein derivative from Mycobacterium bovis (PPD-B), which are bovine Toll-like receptor 4 (TLR4) and TLR2 antagonists, respectively, were also examined. We found that Mφ stimulated with FhepES together with LPS or PPD-B have reduced NO or gamma interferon production, respectively. The ability of FhepES to produce AAMφ was found to be heat labile and partially dependent on glycan residues. A possible role for TLR recognition is discussed.

Co-Infection of Cattle with Fasciola hepatica and Mycobacterium bovis - Immunological Consequences

Fasciola hepatica, the liver fluke, is a common parasite of cattle in much of the world. Previously, we have shown that cattle infected with F. hepatica have altered responsiveness (delayed type hypersensitivity reaction and cytokine responses) to M. bovis BCG infection. We hypothesized that co-infection with F. hepatica would, likewise, alter the immune response of cattle to virulent M. bovis infection, with possible implications for disease diagnosis and disease progression. Our previous work with F. hepatica/M. bovis BCG-infected cattle demonstrated a reduction in interferon (IFN)-gamma responsiveness in co-infected animals. Similar findings are reported here with virulent M. bovis following aerosol infection. The epidemiological significance of these findings, also, require exploration, particularly in view of the considerable resources devoted to the diagnosis and eradication of bovine tuberculosis, and the high prevalence of F. hepatica infection in areas where eradication has proved difficult.

Coordinating innate and adaptive immunity in Fasciola hepatica infection: implications for control.

The helminth parasite Fasciola hepatica is responsible for major economic losses in agriculture throughout temperate regions of the globe. Control measures are heavily reliant on chemotherapy resulting in the emergence of drug resistant parasite populations. Novel control strategies based on vaccination ultimately require a deeper knowledge of host-parasite interactions. Herein we discuss recent advances in the understanding of the immune response to F. hepatica placing them in context with previous knowledge and developments from other model systems. Advances in RNAi and proteomics in the context of helminth research should make target identification and characterisation more rapid. In parallel, integration of these technologies with better immunological understanding will be crucial for future research into F. hepatica control measures.