Background
Many prevalent species of parasitic nematodes - such as
Ascaris lumbricoides, which infects over a billion people [
1], or
Necator americanus, the most geographically widespread of the human hookworms [
2] - migrate through host lungs as larvae. Lung tissue is ruptured as the larvae burst out of the blood vessels to enter the alveolar spaces. Although this process is typically asymptomatic in humans, it can also be associated with acute respiratory distress or longer term complications [
3]. For example, infection with lung-migrating helminths has been associated with bronchial hyper-reactivity and other asthma symptoms among children in China [
4] and Brazil [
5].
The rodent parasite
Nippostrongylus brasiliensis (
Nb) has proven a valuable laboratory model for nematode migration through the host body. In mice, L3 larvae injected into the skin migrate via the lungs to the small intestine, where the parasites develop into adults [
6]. Peak abundance of
Nb larvae in the lung occurs around 2 days post-infection (pi) in many strains of mice [
7]. The lung migratory stage of
Nb is associated with a strong local Type 2 inflammatory response that includes T-helper (Th)2 cells, eosinophils and basophils [
8,
9]. Alternatively-activated macrophages (AAMφ) have also been identified as a major component of the pulmonary response to
Nb infection [
10,
11]. AAMφ are characterised by IL-4/IL-13-dependent production of chitinase and Fizz/resistin family members (ChaFFs) including RELMα (also known as Fizz1), the chitinase-like protein Ym1, and Arginase-1 [
12‐
15], and all three proteins are consistently observed in the
Nb infected lung [
10,
11,
16‐
18]. Arginase-1 is the counter-regulatory enzyme to iNOS and can thus act to suppress NO production and Type 1 effector function. Arginase-1 also has well documented roles in tissue repair [
19,
20] and has recently been implicated as an anti-nematode effector molecule [
21]. The functions of RELMα and Ym1 are less well understood but, like Arginase-1, they have been strongly implicated in the response to injury [
22‐
24] and have putative roles in the repair process, including extra-cellular matrix deposition and angiogenesis [
25,
26]. However, recent data have shown that RELMα and macrophage-derived arginase can also negatively regulate Th2 effector responses and thus limit the pathology associated with overzealous repair [
27‐
29].
Although not formally proven, the association of Arginase-1, RELMα, and Ym1 with the tissue repair process suggests that in the context of nematode infection, ChaFFs, potentially produced by AAMφ, may be required to orchestrate the repair of damage caused by larval migration in order to restore lung integrity. Two recent papers have highlighted the potential for
Nb migration to damage the lung with potentially long term consequences [
16,
18]. Both studies document haemorrhaging of lung tissue and sustained increases in airway hyper-responsiveness. A striking novel observation in these studies is that
Nb causes disruption of the alveolar architecture that is consistent with pulmonary emphysema many weeks after infection. Dysregulated, AAMφ-mediated repair of the damage caused by the nematodes may be responsible for such detrimental outcomes [
16].
Helminths with lung migratory stages are often co-endemic with Type 1-inducing parasites such as malaria [
30‐
32]. Given the potential for cross-regulation between Type 1 and Type 2 immune responses, we wished to use mouse models to investigate the consequences of co-infection for the pulmonary Type 2 immune responses induced by nematode migration. We chose to focus on
Nb and a rodent malaria,
Plasmodium chabaudi chabaudi (
Pcc), that induces a potent Type 1 immune response and non-lethal infection [
33]. We challenged hosts simultaneously with these two acute infections, thus demanding polarized, conflicting immune responses at the same point in time. In addition, we expected
Nb-Pcc co-infection to induce conflicting responses in the same anatomical location, because malaria-infected red blood cells (RBCs) of many species, including
Pcc, adhere to endothelial cells of the microvasculature of the lung [
34‐
36]. Furthermore, malaria itself has been shown to cause lung injury [
37,
38]. Thus, we expected the lung and draining (thoracic) lymph nodes to be potential sites of strong interactions between
Nb and
Pcc. The idea that helminth-malaria co-infection may impose Type 1-Type 2 immunological conflict is not new [
30], nor is the idea that parasitic co-infection may alter the severity of pulmonary disease [
39,
40], but our emphasis on the consequences of malaria for pulmonary Type 2 responses has not previously been explored.
Using these model systems, we assessed production of the ChaFFs, RELMα and Ym1 as primary read-outs of the Type 2 effector response in the lung. We also examined thoracic lymph node (TLN) cytokine profiles, parasitology and systemic pathology, to set the co-infected lung in its whole-organism context. By 7 days pi, malaria infection had significantly reduced the expression of ChaFFs in the lungs of co-infected animals relative to those with
Nb only. This reduction correlated with changes in Th2 cytokines in the TLN, with co-infected mice producing significantly less IL-13, IL-10 and IL-5 than mice infected with
Nb only.
Pcc co-infection thus reduced the extent of pulmonary Type 2 activation and Th2 polarisation in response to
Nb. Future long-term experiments (up to a year in duration [
16]) in the co-infection model established here will explore how helminth migration may interact with malaria infection to affect chronic lung pathology.
Discussion
Our primary aim in this study was to address the interplay of two acute infections that place conflicting demands on the host immune response, particularly in the lung. We wanted to focus on Type 1-Type 2 cross-regulation rather than any effects of regulatory T cells, so we opted for a model of acute rather than chronic helminthiasis. In addition, although anatomical compartmentalization does not preclude immunological interaction - for example, gut-restricted helminths can induce a strong systemic Th2 bias [
47] - compartmentalization can buffer the effects of co-infection [
48]. We thus chose murine infection models that would pose an immunological conflict in anatomical space as well as time post-infection (pi). The dynamics we were investigating may have real life corollaries, because nematode migration occurs in the lungs of over a third of the world's human population [
1,
2], many of whom are co-infected with malaria [
31,
32]. However, because both of our murine models (
Pcc and
Nb) produce self-resolving infections, the effects of co-infection on anaemia and on pulmonary immunology reported here cannot be firmly associated with chronic disease outcomes until longer term co-infection studies are performed.
We quantified the health of mice in our experiments using two measures that have proven informative during
Pcc infection [
49] and
Pcc-nematode co-infection [
50]: body mass and RBC density. To our knowledge, this is the first report demonstrating that murine
Nb infection has a negative impact on both parameters, although reduced weight gain in young
Nb-infected mice has previously been reported [
40].
Nb caused a statistically significant, transient ~3% loss of body mass from approximately 2-4 days pi, and in other experiments using a higher dose (500 L3s), mice lost closer to 10% of their starting body mass (unpublished data). Migration of
Nb larvae through the lungs has previously been shown to cause two spells of inappetance and thus weight loss in rats, one associated with migration of larvae and the other with establishment of adults in the gut [
41]. We detected only one period of loss of body mass; mice may be spared the second spell given the brief survival of adult
Nb in mice, particularly for parasite strains, such as ours, that are not mouse-adapted. We also observed a transient loss of RBC density in
Nb-infected mice. It was rather surprising that this effect - most likely caused by haemorrhaging of the lung following larval migration - was detectable at the systemic level. This suggests that the capillary damage and ingestion of RBCs by alveolar macrophages following lung migration of
Nb [
11,
16] are associated with considerable blood loss.
A diverse range of outcomes is possible when helminths and malaria co-infect a host. Co-infected mice in our study experienced two periods of RBC loss in quick succession - first
Nb-induced and then
Pcc-induced. However, they had slightly higher RBC densities than
Pcc-infected mice did, at the time of most severe malarial disease. This was associated with a small reduction in malaria parasitaemia in the blood. These results contrast with several studies of helminth-malaria co-infection in mice, in which malaria parasitemia was increased [
51‐
54], and/or malarial symptoms exacerbated, in at least some groups of co-infected mice [
50‐
55]. For example, in contrast to the lethal inflammatory liver disease recently described in mice simultaneously co-infected with
Heligmosomoides polygyrus and
Pcc [
55], we observed subtle amelioration of malarial disease and no deaths. This disparity in the severity of co-infection could be due to the fact that we worked with a different mouse strain (BALB/c versus Helmby's C57BL/6) as well as a different helminth species that migrates differently through the host body. However, we detected an elevation in MSP-1
19-specific IL-6 due to co-infection, so it is possible that the emergent IL-17/IL-23 axis described by Helmby [
55] may likewise be involved in our co-infection system, though not in organs that negatively impact short-term survival. Indeed, the mechanisms underlying the slightly protective effect of
Nb observed here are not yet clear. We are investigating possible immunological causes of this protection, including innate mechanisms such as IFN-γ
+ NK cells [
56] and adaptive mechanisms such as cytophilic antibody isotypes [
33] that could promote malaria clearance; either might be altered by acute
Nb co-infection. However, it is also possible that lower parasitaemia might be the consequence of the RBC density changes induced by
Nb, as previous co-infection studies have shown that helminths can limit RBC availability to malarial parasites and thereby cap their replication (e.g., [
57]). Control of microparasites by Th1 immunity and by RBC limitation are not mutually-exclusive possibilities [
58] and both might be operating in our model system. Finally, it is possible that the sequestration habits of
Pcc parasites [
59] are altered by
Nb. These mechanisms remain to be investigated.
Our results largely resemble those reported for other
Nb-microparasite pairings. For example, during co-infection of mice with
Nb and either
Toxoplasma gondii [
60] or
Chlamydophila abortus [
61], significantly reduced Th2 responses (compared to mice with
Nb infection) have been observed, independent of the interval between infections [
60,
61]. These data suggest that
Pcc is not the only microparasite that might reduce Th2 responses to
Nb infection.
Mycobacterium bovis BCG co-infection, however, does not significantly impact
Nb-induced IL-4 in the mesenteric lymph nodes [
62]; it would be of interest to know whether those lung-dwelling microparasites might have had similar effects to
Pcc on Th2 responses, had they been measured in the TLN. Reported effects of
Nb on the course of microparasite infections are likewise mixed: densities of
T. gondii [
60] and
M. bovis BCG [
62] are unaffected by the presence of the nematode, while
C. abortus density increases dramatically [
61]. Interestingly, the concurrent presence of influenza virus with migrating
Nb larvae in the lung exacerbates the severity of lung disease compared to mice with influenza alone [
40]. A two-week delay between
Nb infection and influenza infection, or replacement of
Nb with
H. polygyrus, eliminates the added pathology, suggesting that the simultaneous presence of larvae and virus in the lungs is required [
40]. Such may also be the case for
Pcc-Nb co-infection. The
Nb-influenza study did not include immunological measurements, so the role of the immune system in generating the observed pattern is not known. Indeed, this comparison illustrates that many details of anatomical location and parasite life cycles, as well as immunological interactions, must be taken into account to explain the diverse outcomes of helminth-microparasite co-infections [
30,
46].
Our most novel finding is that malaria infection has the capacity to modulate the host's pulmonary Type 2 response to nematode migration. However, the long-term impact of the altered Type 2 response is not possible to predict, because the function of Type 2 immunity in this setting is not yet fully understood. There are at least three potential outcomes of a helminth-induced Type 2 response in the lung. First, it may contribute to protection against incoming larvae [
6]. Second, Type 2 responses are likely to be involved in repairing the damage that is inflicted by migrating parasites. Third, as recent studies have shown [
5,
16,
18], lung migration and the associated Th2 responses have the potential to cause long-term lung pathology. Appropriate repair versus lung malfunction are likely to be flip sides of the same coin. Indeed, although ChaFFs and Arginase-1 are implicated in tissue repair, they are also associated with fibrosis, an overzealous repair process [
19,
24,
43,
63‐
65] (see also review by Wynn [
66]). Predicting the effect of
Pcc co-infection on long term
Nb-induced lung disease is further complicated by recent data that suggest both Arginase-1 and RELMα can negatively regulate Th2-mediated pathology [
27‐
29]. By this logic, inhibition of these molecules by malaria co-infection may ultimately exacerbate Th2-mediated lung damage.
However, our data suggest that the effect of malaria on ChaFF expression is not direct but rather via reduced Th2 cytokines. The effect of
Pcc on
Nb-induced ChaFFs was not apparent until 7 days pi, when the extent of the increase in ChaFF expression was inhibited by co-infection. This was correlated with differences in cytokine production in lymphocyte recall assays, suggesting that changes in ChaFF expression were driven by changes in the T lymphocyte populations after the onset of the adaptive Th2 immune response (around 5 days pi, as shown in
Nb- infected IL-4 reporter mice [
9]). A role for adaptive immunity is further supported by work showing that SCID mice are not able to sustain AAMφ responses in the lung following
Nb infection [
11], and a demonstrated requirement for T cells to sustain the AAMφ response in a mouse peritoneal infection model [
24]. Remarkably, in SCID mice, in the absence of T cells and AAMφ, the
Nb-induced cellular infiltrate does not resolve [
11]. The capacity of malaria to inhibit the transition to a full Th2 response by 7 days pi may likewise be detrimental to full resolution of the inflammatory response, a step necessary for appropriate tissue repair [
67,
68]. By day 20 pi, however, the residual Th2 responses in co-infected mice were as high as, or even higher than, in
Nb-only mice. In support of this, day 20 antigen-specific IL-5 responses were particularly high in co-infected animals. Thus
Pcc infection may protect against airway hyper-responsiveness through a reduction in peak Th2 activation, or else exacerbate it due to sustained Th2 activity. Transient passage of
Nb larvae through the lung inflicts lasting damage [
16,
18]. Whether transient impairment of pulmonary Th2 responses by malaria co-infection also has lasting effects needs to be investigated experimentally.
A perhaps surprising finding in our study was the apparent absence of classical macrophage activation in the lung despite the clear presence of malaria parasites: we did not detect iNOS, IL-12p40 nor elevated TNF-α mRNA in lung tissue of
Pcc-only or co-infected mice at any time point. One could argue that malaria parasites stay in the lung microvasculature and do not cross into tissue. However, this is unlikely to be the case, given the extensive lung damage due to
Nb in co-infected mice, as well as evidence that malaria merozoites can be found dispersed in the lung [
34]. Furthermore, IFN-γ mRNA was detectable in the lung of all
Pcc mice regardless of co-infection, suggesting that lymphocytes were activated, perhaps by innate activation of NK or γδ T cells. The most likely explanation for the failure to detect classical macrophage activation may be that lung macrophages, which are exposed daily to inhaled microbes, have a remarkably high threshold for activation even in the presence of IFN-γ and microbial stimuli [
69].
As with any laboratory model, it is important to acknowledge the potential disconnection between natural co-infections and the experimental systems and designs used here, including the relative timing of the two infections, doses at which they were administered, and the fact that we have only studied primary and self-resolving (rather than secondary and/or chronic) infections. Permutation of any of these parameters is likely to quantitatively, if not qualitatively, alter outcomes. For example, repair processes might readily keep pace with lung damage when the rate of exposure to nematode larvae is low, unlike in most experimental models. We used a relatively low dose of L3 larvae (200 per mouse while others use ~500 [
16,
18] or as many as 750-1000 [
60‐
62]) but still exceeded natural exposure levels. Furthermore, larval helminths and malaria parasites are unlikely to arrive in the lung within a few days of each other in nature, and it may be that pre-existing malaria would have had a different effect on pulmonary Type 2 responses to
Nb migration, particularly if malaria parasites do not remain long in the lung. Indeed, the most likely natural exposure scenario may be chronic malaria infection into which helminth larvae are "trickled" [
32], but experimental studies that mimic this scenario have yet to be carried out. Nonetheless, lung dysfunction is seen as a consequence of helminth migration [
4,
5] and both acute and persistent malaria infection [
38] in people, so high-dose experimental
Nb studies in which long-term lung pathology can be observed [
16,
18], combined with simultaneous malaria exposure, may provide useful models for disease states in people.
Authors' contributions
MAH conducted RT-PCR analysis of ChaFFs and PCR analysis of Pcc genomes, ran Western blots, statistically analysed some of the data, assisted with IHC scoring, and helped to draft the manuscript. KJM led the lung sampling, conducted cytokine RT-PCR reactions, assisted with Western blots, did IHC staining and scoring, and helped to draft the manuscript. Additionally, KJM collected all data for the Nb timecourse experiment. KJF-C, aided by ALG, set up all co-infection experiments and collected parasitemia, body mass and anaemia data. SM, aided by KJF-C, cultured lymph node cells and measured cytokines and cytokine receptors in the supernatants. JEA and ALG conceived of and designed the study, and drafted the manuscript. ALG performed most of the statistical analysis. All authors contributed to scientific discussions of the data, read and approved the final manuscript.