Current Research

 

Projects:

  1. Macrophages (M φ) in Helminth Infection: Effectors, Regulators, or Healers?
  2. Vaccination against filariasis and cytokine mediated host-parasite dynamics
  3. Chitinases: New players in Th2 mediated allergic lung inflammation
  4. Co-infection with parasites that elicit type 1 vs. type 2 immune responses
  5. Innate macrophage activation by the surface mucins of Echinococcus granulosus

Funding

Funding for this work is provided by:

the MRC

MRC: Medical Research Council

 

the Wellcome Trust

the Wellcome Trust

the BBSRC

the European Union

the European Community

Asthma UK

Macrophages (Mφ) in Helminth Infection: Effectors, Regulators, or Healers?

Macrophages are recruited in large numbers to the site of helminth infection and yet their function in these settings has been poorly understood.  Using an infection model system with the human parasite, Brugia malayi we are able to generate large numbers of macrophages in a potent type 2 environment (altenatively-activated Mφ) and thus have a unique opportunity to investigate this cell type in vivo.  We have found that nematode elicited macrophages (NeMφs, pronounced “NeeMacs”) can actively block the proliferation of a broad range of cell types (Loke 2000a). In addition, these cells are characterized by the extremely high level expression of two novel proteins of unknown function, Ym1 and RELMa (Loke 2002).  Using several model systems available in the laboratory, we demonstrated that this distinct macrophage phenotype is a generalised feature of nematode infection (Nair 2005). It is now apparent that the phenotype we describe is characteristic of macrophages in a broad range of Th2 mediated chronic inflammatory settings (Rodriguez-Sosa 2002, Raes G 2001). Details on EST analysis of NeMφs available at this site.

As part of a MRC funded project, we are working to elucidate the function of alternatively-activated Mφ in helminth settings and addressing the following questions.

Effector Function: Th2-mediated immunity, dependent upon IL-4/IL-13, is typically associated with IgE, eosinophils, mast cells and goblet cells. Until recently a role for Mφ had been largely ignored despite their accumulation in large numbers at the site of helminth infection. Although it is now appreciated that IL-4/IL-13-responsive Mφ (alternatively activated Mφ) have a distinctive phenotype (Loke 2002) the possibility that they act against certain extracellular pathogens, analogous to classically activated Mφ and intracellular pathogens, has yet to be fully addressed. Litomosoides sigmodontis is a filarial nematode parasite that, uniquely, undergoes full development from infective larvae through to blood-circulating microfilariae in inbred laboratory mice (Allen 2008).  This provides a dynamic model system to study effector function at successive stages of infection with a tissue-dwelling nematode. The possibility that IL-4/IL-13 activated AAMφ act as effector cells during filarial infection is supported by our own and other studies showing that IL-4-dependent mechanisms mediate resistance (LeGoff 2002) and that parasite killing occurs in the presence of AAMφ (Taylor 2006).

Regulation (in collaboration with Rick Maizels & Matt Taylor).  Immune down-regulation is a cardinal feature of helminth infection and has been extensively documented in both human disease and animal models.  A remarkable finding has been the loss of antigen-specific in vitro T cell responses in infection, because it correlates both with reduced pathology and a failure of the immune system to kill the parasite.  Work with L. sigmodontis has directly addressed this issue. During infection, three distinct factors contribute to impaired T-cell responses: Mφ that block cellular proliferation, suppressive T regulatory (Treg) cells and hypo-responsive effector T cells (Taylor 2005, Taylor 2006). Following Treg depletion, the AAMφ population retains its full capacity to suppress cellular proliferation (Taylor 2006) suggesting that their development is not contingent upon Treg cells.  Conversely, however, AAMφ may be important for the induction of Treg cell activity.  We are investigating the contribution of Mφ to the regulatory environment produced during helminth infection.   Determining when and where NeMφ function as regulatory cells vs. effector cells is a key focus of studies in this project.

Healing: The damaging profibrotic aspects of IL-13, some of which are mediated by AAMφ, are now well recognized (Wynn Nat Rev Immunol 2004). However, we and others have argued that AAMφ must also have more appropriate roles in tissue remodeling and wound healing. For example, the 3 most abundant proteins produced by nematode-induced Mf; Ym1, RELMa and Arg1 are induced in response to injury even in the absence of a Th2-inducing parasite, implicating these proteins in the response to tissue injury (Loke 2007).  A question raised by the association of Th2 responses with tissue repair, is why helminth parasites induce an immune response that has wound repair as a primary function. We have proposed that the propensity of many parasites to induce potentially lethal tissue damage provides sufficient evolutionary pressure for the development of a worm-specific tissue repair process (Graham 2005). A dramatic example of repair is seen during N. brasiliensis infection, where larval migration causes severe pulmonary damage with leakage of serum proteins into the lavage fluid. However, by two weeks the lung has undergone a remarkable level of repair. Maximal Th2 responses occur well after the parasite has left the lung and Ym1/RELMa levels in the lungs of N. brasiliensis infected mice continue to increase for over 2 weeks (Nair 2005). We are using this as a model to investigate the hypothesis that Th2 immune responses in the lung have a predominant role in repair or ‘clean up’.

Beyond IL4 and IL-13: what determines Mφ activation during chronic infection?Nematode-elicited Mφ can be described as alternatively-activated because of their dependency on IL-4/IL-13with regard to anti-proliferative capacity(Loke 2000), Ym1/RELMa/Arg1 expresssion (Loke 2002, Nair 2005) and reduced pro-inflammatory chemokines (Loke 2002).  However, in the context of infection, Mφ will be exposed to many additional signals that may modulate or enhance these functions. Consistent with this we observe Mφ phenotypic changes during nematode infection that are not lost in the absence of IL-4/IL-13 even in the face of a switch to a more classically-activated Mφ phenotype. These include an enhanced ability to engulf phagocytic cells(Loke 2007), a rounded cell shape(Nair 2003), and IL-10/TGF-b production (Loke 2000, 2007). NeMφ will be exposed to a range of host and parasite factors that may contribute to their functional phenotype. We hope to delineate the signals beyond IL-4 and IL-13 that will determine macrophage-dependent outcomes during helminth infection.

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Vaccination against filariasis and cytokine mediated host-parasite dynamics

(in collaboration with David Taylor )

Work in filariasis was constrained by the absence of a laboratory model to investigate both the immunology and the natural migration of filarial parasites. The discovery by Odile Bain’s group in Paris that Litomosoides sigmodontis can produce patent infections in the BALB/c strain has removed this impediment. Entering at the mite vector bite site, L. sigmodontis vector-derived larvae (L3) migrate via the lymphatics, reaching the thoracic cavity by 6 days post-infection. There they mature and by day 55 microfilariae (MF) are found in peripheral blood. In contrast, on the C57BL/6 background the parasite numbers decline steadily over time and the parasites never reach sexual maturity (Graham 2005b). Using this model system it has become possible to ask: What is the role of the Th2 mediated response in resistance to filarial infection (LeGoff 2002) and what is its role at different stages of the parasite life cycle? Although the data initially seemed to build a convincing picture of Th2 responses in the control of filarial infections, the real picture that is emerging is far more complex with different stages controlled by different cytokines. Additionally, T regulatory cells can override effector function in susceptible strains (Taylor 2005). Despite our increasing understanding of the cytokine pathways that determine infection outcome, we are still remarkably ignorant about the exact mechanisms by which these large multicellular parasites are destroyed. What are the physical means (cells, antibodies, other?) enabled by cytokines that lead to parasite death?

These questions are relevant to efforts to develop vaccines against helminth parasites. The ability of irradiated L3 to generate protection has long since been demonstrated in a variety of helminth systems. Immune protection generated by irradiated L. sigmodontis larvae leads to rapid destruction of the challenge larvae in the subcutaneous tissue (LeGoff 1997) and is long lived (Babayan 2006) dependent on the type 2 cytokine IL-5 (LeGoff 2000). However, how irradiated larvae induce this protective response is not known. Irradiated larvae may be failing to shut down the expression of early genes and thus potentially over-expressing immunogenic molecules. Conversely (but not mutually exclusive) irradiated L3 may be failing to produce molecules that initiate down-regulatory pathways in the host. We are also actively involved in anti-filarial vaccine studies designed to promote Th2 response through the use of DNA vaccines that express L3-specific genes (Allen 2000). An EST project in collaboration with Mark Blaxter's lab provides a valuable resource for these studies.

Collaborators on this project: Odile Bain, Museum of Natural History Paris and David Taylor, University of Edinburgh. This work is funded by the European Union as part of a programme entitled "SCOOTT: sustainable control of onchocerchiasis today and tomorrow".

Simon Babayan as part of a Marie Curie fellowship has elucidated a role for IL-5 as a developmental cue utilised by Litomosoides sigmodontis and is investigating the evolutionary and mechanistic impact of these findings. Simon is also collaborating with Jos Houdijk of the Scottish Agricultural College, to investigate the role of nutrition on immunity to nematodes.

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Chitinases: New players in Th2 mediated allergic lung inflammation

(in collaboration with Rick Maizels)

Our interest in mammalian chitinases arose when we discovered that the chitinase family member Ym1 was the most abundant protein secreted by macrophages activated under Th2 cytokine conditions in vivo (Loke 2002) .  Subsequently, we and others demonstrated that Ym1 can be induced directly by in vitro treatment of macrophages with IL-4 or IL-13 (Nair 2003) and expression of Ym1 is abolished when both IL-4 and IL-13 signalling are absent.  In the course of our investigations we discovered that in addition to Ym1, acidic mammalian chitinase (AMCase) was produced during Th2 mediated inflammation in the lung (Nair 2005). Ym1 and AMCase are closely related by sequence homology with similar sugar-binding properties, but differ in that only AMCase has functional chitin cleaving activity11,12. A near-identical form of Ym1, termed Ym2, is also expressed.

AMCase, under the control of the Th2 cytokine IL-13 has been strongly implicated in asthma-related pathology (Zhu Science 2004).  However, Ym1, the inactive chitinase family member, is also expressed at very high levels in the lung in mouse models of allergic asthma and that expression of both AMCase and Ym proteins are dependent on IL-4 and IL-13. The hypothesis we are testing is that these two chitinase family members have overlapping but distinct functions that are essential components of lung pathology during allergic responses. This work is supported by AsthmaUK

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Co-infection with parasites that elicit type 1 vs. type 2 immune responses

(in collaboration with Andrea Graham, Karen Grocock & David Gray)

The evidence that helminth infection can alter immune responsiveness to a second infection is irrefutable. A more important issue is how this modulation influences the host in terms of reduced or increased immune pathology and parasite intensity. Not surprisingly, the answers differ radically depending on which pathogens are studied and the species of co-infected host. Our work in collaboration with Andrea Graham has elucidated an intriguing relationship in malaria-filaria co-infection (Graham 2005a). We found that co-infection significantly enhanced the severity of malaria but only among hosts with no circulating microfilaria (MF-). The data strongly suggested that the penalty of co-infection did not occur in MF+ hosts because they were able to downregulate the more severe consequences of the pro-inflammatory response. We have also investigated the consequence of a pre-existing helminth infection on Leishmaniasis and found that despite highly compartmentalised and appropriately polarised responses to each parasite, helminth infection was still sufficient to delay leishmania-induced lesion progression(Lamb 2005).

A common theme in many helminth co-infection studies is a downregulated pro-inflammatory response. This may have two very different consequences for the host: a beneficial reduction in immunopathology or conversely, disease exacerbation due to inadequate control of parasite replication. One of the many challenges to co-infection research is to distinguish the effects of co-infection on parasite control from immunopathology. In collaboration with Andrea Graham, we are now investigating co-infection of malaria with the gastrointestinal helminth, Nippostrongylus brasielinsis. Because Nb can cause significant damage while migrating through the lung and P chabaudi (our model malaria) sequesters in the lung, we are able to address questions about how the immune systems copes with two parasites that meet head on but make differing demands on the host.

In collaboration with David Gray, we are also investigating the impact of co-infection on the memory response. We are examining sequential infections with type 1 vs. type 2 parasites to ask whether the memory response is committed to a particular cytokine pathway. Our co-infection studies also overlap with our interest in macrophages as we are addressing the ability of macrophages driven to an alternatively-activated phenotype to cope with intracellular pathogens.

This work is funded by the BBSRC.

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Innate macrophage activation by surface mucins of Echinoccocus granulosis

(in collaboration Alvaro Diaz, Montevideo, Uruguay)

Immune responses to parasites differ not only along the type 1-type 2 axis but also along an inflammatory-regulatory, axis (Diaz, 2007). Thus, while allergies are type 2 and inflammatory, typical responses to worms are type 2 and regulatory, a combination known as the “modified type 2” response (Nat Rev Immunol 3:733). All immune responses are ultimately defined by the way in which microbial components are decoded by innate immune receptors. For modified type 2 responses, the pathogen molecules and innate receptors that start the signaling process are largely unknown but carbohydrates and their lectin receptors may be crucial players. The larval E. granulosus model has outstanding features for studying innate activation by parasitic worms. This cestode elicits a type 2-biased and anti-inflammatory response. The efficacy of the inflammatory control is remarkable given the years-long permanence of the large bladder-like larvae in host internal organs. In addition, the major molecules exposed by the parasite, including probable immunomodulators, are available in unusually large amounts, and their structural elucidation is under way in the Diaz lab. These are mucins (heavily glycosylated polypeptides) that form an insoluble meshwork. Cecilia, a PhD student in Alvaro Diaz’ lab will making 3 several month visits to the Allen lab to investigate the impact of the of E. granulosus mucins on the activation state of macrophages.  Cecilia is supported by the British Council and the Royal Society. 

 

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