Innate Immune Responses to Lung-Stage Helminth Infection Induce Altern
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《感染与免疫杂志》
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland
ABSTRACT
While it is well established that infection with the rodent hookworm Nippostrongylus brasiliensis induces a strongly polarized Th2 immune response, little is known about the innate host-parasite interactions that lead to the development of this robust Th2 immunity. We exploited the transient pulmonary phase of N. brasiliensis development to study the innate immune responses induced by this helminth parasite in wild-type (WT) and severe-combined immune deficient (SCID) BALB/c mice. Histological analysis demonstrated that the cellular infiltrates caused by N. brasiliensis transit through the lungs were quickly resolved in WT mice but not in SCID mice. Microarray-based gene expression analysis demonstrated that there was a rapid induction of genes encoding molecules that participate in innate immunity and in repair/remodeling during days 2 to 4 postinfection in the lungs of WT and SCID mice. Of particular note was the rapid upregulation in both WT and SCID mice of the genes encoding YM1, FIZZ1, and Arg1, indicating a role for alternatively activated macrophages (AAMs) in pulmonary innate immunity. Immunohistochemistry revealed that nearly all alveolar macrophages became YM1-producing AAMs as early as day 2 postinfection. While the innate responses induced during the lung phase of N. brasiliensis infection were similar in complexity and magnitude in WT and SCID mice, only mice with functional T cells were capable of maintaining elevated levels of gene expression beyond the innate window of reactivity. The induction of alternatively activated alveolar macrophages could be important for dampening the level of inflammation in the lungs and contribute to the long-term decrease in pulmonary inflammation that has been associated with helminth infections.
INTRODUCTION
The cells and molecules that comprise the innate immune responses are the first line of defense against invading pathogenic organisms. In addition, innate immune mechanisms are important for the elimination of the myriad of nonpathogenic substances to which we are continuously exposed. In those circumstances in which the effector mechanisms of the innate response are not sufficient to fully eliminate a pathogen challenge, the same cells and molecules function to efficiently activate the antigen-specific adaptive arm of the immune response. In recent years, it has become clear that different modes of activation of innate immunity have a profound influence on the magnitude and the nature of subsequent adaptive responses (9, 32, 69).
Parasitic nematodes are among the strongest natural inducers of polarized Th2 immune responses (2, 40, 60). Infection of mice with the intestinal nematode parasite Nippostrongylus brasiliensis has been used extensively to study the regulation of immunoglobulin E synthesis (31) and Th2 immunity in general (19, 33). Despite the longstanding use of N. brasiliensis as a model, little is known about the innate immune responses that precede the induction of a highly polarized adaptive Th2 response to N. brasiliensis challenge. Indeed, there are only limited data on the dynamic interplay between helminth antigens and the receptors of the innate immune system of mammals (1, 3, 21, 68). Given the exceedingly high prevalence of helminth infections in human and animal populations (6, 11) and the recent appreciation that helminth infections influence subsequent responses to other pathogens (65), environmental antigens (76), and vaccines (10, 52), defining the innate-mediated responses induced by helminth parasites will provide a basis for understanding the downstream cellular and molecular events that lead to highly regulated immune responses.
In addition to being a tested model for the study of Th2 immune responses, N. brasiliensis infection in mice serves as a model for human hookworm infections with Necator americanus and Ancylostoma duodenale. Human and rodent hookworms, as well as a number of other helminth species of public health importance, migrate through the lungs of the host during larval development. This obligate pulmonary phase of the life cycle typically occurs within a few hours after infection and lasts only about a day before the worms migrate to the intestine, where they develop into adults. The significance of this transient migration through the lungs to the physiology of the parasite is not clear. However, one of the consequences of this brief exposure on the host is a substantive, prolonged alteration of the immunological status of the lung. For example, the level of allergen-induced pulmonary inflammation was significantly less in mice that were previously infected with N. brasiliensis than in noninfected controls (42). The results derived from the N. brasiliensis-mouse model are consistent with the inverse correlations made in human populations between helminth infection and the prevalence of allergic asthma (43, 79).
To gain insight into the mechanisms underlying parasite-induced alterations in immunological responsiveness, we examined the lung phase of N. brasiliensis development in wild-type (WT) and severe-combined immune deficient (SCID) mice. SCID mice were employed to study innate responses in an environment devoid of the major cellular mechanism necessary to mount an adaptive immune response. N. brasiliensis induced a rapid and robust innate immune response in the lungs of both WT and SCID mice. Expression profiling showed that both strains of mice transcribed a similar subset of genes during the innate response to N. brasiliensis infection. Of particular note was the strong expression of genes associated with alternatively activated macrophages: ym1, fizz1, and arg1. The presence of alternatively activated macrophages (AAMs) in the lungs of both WT and SCID animals was confirmed by immunohistocytochemistry. Although the initial responses to N. brasiliensis were similar in complexity and magnitude in the lungs of WT and SCID mice, only WT mice were capable of maintaining elevated levels of gene expression beyond the innate window of reactivity. These results provide novel insights into the cellular and molecular basis for infection-induced modulation of pulmonary immune reactivity.
MATERIALS AND METHODS
Animals and infection. Six- to eight-week-old male WT and SCID mice on a BALB/c background were obtained from the National Cancer Institute at Frederick, Md. Mice were housed in barrier filter-top cages, given food and water ad libitum, and kept on a 12-h light/dark cycle. Infectious third-stage N. brasiliensis larvae were harvested from a fecal culture via a Baermann apparatus, washed in phosphate-buffered saline, and counted. Mice were infected subcutaneously with 500 larvae. All experimental procedures described in this paper were performed under the approval of the Johns Hopkins University Animal Care and Use Committee in accordance with the guidelines set out by the National Research Council's Guide for the Care and Use of Laboratory Animals.
Histology. (i) Lung inflations. Animals were anesthetized with 300 mg/kg of body weight of Avertin (2,2,2-tribromoethanol) administered intraperitoneally. Pericardium and trachea were exposed by dissection. A lateral incision was made through the trachea, and a 19-gauge flat-tipped needle was inserted and tied off. Inflation of the lungs was performed slowly over 2 min with 0.8 ml of zinc-buffered formalin fixative (Z-fix) (Anatech Ltd., Battle Creek, MI). The trachea was then tied off, and the lungs were removed from the pericardial cavity and incubated in a 40-fold volume of Z-fix.
(ii) Histological preparation. Inflated and fixed lungs were embedded in paraffin, and sagittal 10-μm sections were obtained from four different levels of the lung. Sections were stained with hematoxylin and eosin (H&E) or with periodic acid Schiff plus hematoxylin counterstain.
(iii) Immunohistochemistry. Sections were stained for alveolar macrophages using biotinylated Griffonia (Bandeiraea) Simplicifolia lectin 1 (GSL I) (10 μg/ml; Vector Laboratories, Burlingame, CA) followed by incubation with alkaline phosphatase-conjugated streptavidin (1:200) (Vector Laboratories) and Liquid Permanent Red chromogen (DAKO USA, Carpinteria, CA). YM1 staining was performed using affinity-purified goat antimouse YM1 (1 μg/ml; R&D Systems, Minneapolis, MN). Anti-YM1 was detected using a biotinylated rabbit antigoat immunoglobulin G (Vector Laboratories), peroxidase streptavidin and 3,3'-diaminobenzidine (Vector Laboratories). For dual-fluorescence staining of macrophages, YM1 was resolved with Fluorescein/Avidin DCS (Vector Laboratories) and, after an avidin/biotin blocking step (Vector Laboratories), GSL I binding was detected with Texas Red/Avidin DCS. Stained sections were mounted in Vectashield containing 4'-6'-diamidino-2-phenylindole (Vector Laboratories).
(iv) Enumeration. The values from three WT and three SCID animals at 0, 2, 4, 8, and 12 days postinfection were used to evaluate the changes in alveolar macrophage and AAM numbers. The GSL I-positive and YM1-positive cells were counted in each of six contiguous lung histological fields (magnification, x10; 974 mm2/field), and the mean cell number per field was calculated. Statistical significance was determined using a two-tailed paired Student t test.
(v) Light microscopy and imaging. Lung sections were examined using a Nikon Eclipse E800 light microscope (Nikon, Inc., Melville, NY), and images were acquired using a SPOT RT charge-coupled-device imager and software (Diagnostic Instruments, Inc., Sterling Heights, MI).
Gene expression analysis. (i) Total RNA extraction. Lungs were harvested, flash frozen in liquid nitrogen, and stored at –80°C. Lungs were homogenized in 2 ml Trizol (Invitrogen, Carlsbad, CA), and 1 ml of homogenate was processed for RNA isolation according to the manufacturer's (Invitrogen) protocol with minor modifications including the use of glycogen at 10 μg/ml as a carrier for an overnight isopropanol precipitation and increasing centrifugation times to 15 min. RNA pellets were resuspended in nuclease-free water. RNA quality was determined by RNA Nano LabChip analysis on a Bioanalyzer 2100 (Agilent, Palo Alto, CA). An RNeasy total RNA cleanup protocol (QIAGEN) was performed, followed by spectrophotometric assessment of the RNA concentration. The lungs from three animals were independently processed for each treatment group and each time point.
(ii) Affymetrix GeneChip protocols. Processing of templates and hybridization for the 430 2.0 array GeneChip (Affymetrix, Inc, Santa Clara, CA) was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision 3, as previously described (30). Following hybridization, the GeneChips were washed and stained in an automated fluidics station (Affymetrix FS450) and then assessed using the GCS3000 laser scanner (Affymetrix) at an emission wavelength of 570 nm at a 2.5-μm resolution. The intensity of hybridization for each probe pair was computed by GCOS 1.2 software (Affymetrix). (For more-detailed methods, please refer to the website of the Malaria Research Institute Gene Array Core Facility at the Johns Hopkins Bloomberg School of Public Health [http://jhmmi.jhsph.edu]).
(iii) Data analysis. Affymetrix CEL file data were preprocessed for use with the probe level GeneChip Robust Multi-Array analysis (77) option in GeneSpring 7 Software (Agilent Technologies). Initial filtering by probe intensity for raw levels above 150 in at least 2 out of 12 conditions resulted in a list of 12,036 genes that were then used as the basis for selecting differentially regulated genes. Raw intensity values ranged from 150 to 46,037.
Real-time RT PCR. From each treatment group and time point, 1 μg of lung RNA was reverse transcribed using the SuperScript first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using an oligo(dT) primer. Resultant cDNAs were amplified for real-time detection with fluorogenic-labeled probes in assays specific for each target gene. Quantitative real-time RT-PCR was performed using the Applied Biosystems 7500 real-time PCR system, TaqMan gene expression assays-on-demand, and TaqMan Universal master mix (Applied Biosystems, Foster City, CA). The following assays (Applied Biosystems product numbers) were used: YM1 (Mm00657889_mH), YM2 (Mm00840870_m1), FIZZ1 (Mm00445109_m1), ARG1 (Mm00475988_m1), interleukin 4 (IL-4) (Mm00445259_m1), IL-13 (Mm00434204_m1), tumor necrosis factor (TNF) (Mm00443258_m1), IL-1 (Mm00434228_m1), IL-10 (Mm00439616_m1), transforming growth factor beta (TGF-) (Mm00441724_m1), IL-6 (Mm00446190_m1), beta-1 interferon (Mm00439546_s1), gamma interferon (IFN-) (Mm00801778_m1), and alpha-2 interferon (Mm00833961_s1). Reactions were performed using 1 μl of cDNA in a 25-μl sample volume and the following thermal cycler profile: 10 min of denaturation at 95°C, 50 cycles of 1-min extension at 60°C and then 15 s of denaturation at 95°C. Analysis was performed using the 7500 system SDS software package (Applied Biosystems).
Microarray data accession number. The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through Gene Expression Omnibus series accession number GSE3414.
RESULTS
Nippostrongylus in the lung. After subcutaneous infection, N. brasiliensis infective-stage larvae entered the general circulation and were carried to the lungs. Between 24 and 48 h postinfection (p.i.), the larvae penetrated alveolar capillaries and transiently took up residence in the alveolar space (Fig. 1I). In both WT and SCID BALB/c mice, focal damage to the alveolar capillaries resulted in hemorrhage into the air space (Fig. 1C and D). By day 4 p.i., the larvae had exited the lungs and entered the small intestine, where they molted to adults, and the females released eggs for 3 to 5 days prior to being expelled from the gut via an immune-mediated mechanism (data not shown) (71). N. brasiliensis was not expelled from the intestine of the SCID mice, since CD4+ T cells are required for resolution of infection (70, 71).
At day 4 p.i. (24 to 36 h after the larvae have exited the lungs), the hemorrhage was largely resolved in WT mice and there was evidence of a modest influx of mononuclear cells (Fig. 1E). In contrast, hemorrhage in the lungs from SCID animals persisted, and the mononuclear cell infiltrate was elevated compared to WT levels at days 4 and 8 p.i. (Fig. 1E through H). In addition to the changes in the parenchyma of the lung, the presence of N. brasiliensis larvae induced epithelial cell and goblet cell hyperplasia (Fig. 1C to H, inserts, and J). The hyperplasia was largely resolved in WT lungs by day 8 p.i. (Fig. 1G) but persisted in the SCID mice (Fig. 1H).
N. brasiliensis larvae induce similar innate responses in the lungs of SCID and WT mice. A gene expression profiling approach was used to study the innate immune response in the lungs during the pulmonary phase of N. brasiliensis infection in WT and SCID BALB/c mice. Lungs from infected animals were removed at days 0, 2, 3, 4, 8, and 12 p.i. and processed for gene expression analysis. Of special interest were genes that were significantly upregulated between days 0 and 4 of infection, corresponding to the period where innate immune responses are dominant. A comparison of the expression profiles in uninfected WT and SCID BALB/c mice showed that just under 7% of the 11,500 genes expressed in the lungs were differentially transcribed in the two strains (data not shown). WT mice differentially transcribed genes associated with adaptive immunity (B- and T-cell-related genes), which were predictably absent in SCID mice. Based on these similarities in expression profiles, gene expression data from N. brasiliensis-infected WT and SCID mice were normalized to the expression levels in strain-matched uninfected lungs.
On day 2 of infection, the newly arrived N. brasiliensis larvae induced at least a twofold increase in the transcription of 148 and 112 genes in the lungs of WT and SCID mice, respectively, compared to the expression profiles of the strain-matched noninfected controls (Fig. 2A). Approximately 35% of the N. brasiliensis-induced genes were common to both WT and SCID lungs at day 2 (Fig. 2A). On days 3 and 4 of infection, an additional 69 and 115 genes were upregulated in WT and SCID animals, respectively, of which 22% were common to both strains. Of the total of 309 different genes whose transcription was significantly augmented by N. brasiliensis at days 2 to 4 of infection in both WT and SCID mice, a direct role in innate immunity as defined by GO slims annotation could be ascribed to approximately one-fourth (76 genes) (see Table S1 in the supplemental material), and 58% (44 genes) of these were induced in both WT and SCID mice. Among the genes upregulated in the lungs of WT (217 genes) and SCID (227 genes) animals on days 2, 3, and 4 of infection (Fig. 2B and C), 72 (33%) and 54 (24%), respectively, had significantly enhanced transcription at all three time points. Over the course of infection, the percentage of genes that concomitantly increased in WT and SCID lungs peaked at 44% at day 4 p.i. and dropped to under 5% at day 12 p.i. (Fig. 2A). By day 12 p.i., only 14 genes were significantly upregulated in the lungs of SCID mice, whereas WT mice maintained an elevated transcription of 78 genes (Fig. 2A; see Table S2 in the supplemental material). Thus, while both WT and SCID mice are capable of rapidly initiating an innate immune response to the migration of N. brasiliensis larvae in the lungs, only WT BALB/c animals are capable of sustaining the response.
With a focus on genes encoding proteins with the potential to participate in innate immune responses, 70 genes had significantly enhanced transcription for at least one time point during the first 4 days of infection of WT or SCID lungs (Fig. 3; Table 1; see Table S3 in the supplemental material). In general, the N. brasiliensis-induced genes showed comparable trends of upregulation in WT and SCID mice (Fig. 3 and 4; Table 1) during the innate window of immune response. In the lungs of SCID mice, many of these genes dropped back to baseline levels after day 4 p.i. (Fig. 3 and 4; Table 1). In contrast, the transcription of most of these genes was maintained or increased in WT mice through day 12 p.i.
Genes encoding proteins associated with allergen-induced Th2 responses in the lungs, such as trefoil factor 2 (TFF-2) (54), small proline-rich protein 2 (SPRR-2) (81), and Gob-5 (80), were also found to be upregulated in the lungs of N. brasiliensis-infected WT and SCID mice (Table 1). Of particular note, transcription of gob-5 was significantly elevated through day 12 p.i. in the lungs of WT animals, whereas gob-5 expression was robust but limited to the innate window in the lungs of SCID mice. Expression of the genes encoding the IL-13- and STAT6-dependent SPRR-2a (81) and TTF-2 (54) were largely restricted to the lungs from WT animals at 8 to 12 days p.i., suggesting that these genes might be under control of adaptive immune mechanisms.
The hypothesis that helminths have evolved mechanisms of evading host defense by suppressing inflammatory responses has been discussed in previous studies (43, 79). The results of our array experiments suggest that downregulation is not a major mechanism during the innate immune response, and helminth-mediated immunomodulation is confined to adaptive immunity. Of the 277 genes that were downregulated more than twofold at any given time point in either WT or SCID mice, less than 7% had an immune-associated function (data not shown).
N. brasiliensis larvae induce repair and remodeling in the lung. The innate inflammatory response induced by larval migration initiated transcription of genes encoding molecules key to lung repair and remodeling, including elastin, procollagen, matrix metalloprotease (MMP12), and cysteine protease (cathepsin K), and extracellular matrix proteins (fibronectin and tenascin C) (Fig. 3; Table 1). In addition, the transcription of the tissue inhibitor of metalloprotease 1 gene was also significantly upregulated, which presumably played a role in limiting the action of MMP12 in the lungs (28). While expression of these genes was induced to comparable levels in the lungs from both WT and SCID mice during the first 4 days of infection, only WT animals were capable of maintaining an elevated level of expression. The results indicate that lymphocytes play a key role in the regulation of a sustained repair process in the lung.
Cytokines and chemokines. IL-4 and IL-13 are key cytokines in the induction and promotion of Th2 immune responses (33). During the innate immune response to N. brasiliensis in the lungs of WT mice, the transcription of the genes encoding IL-4 and IL-13 was variable until day 4 p.i. (Fig. 4B). Between days 4 and 12 p.i., the expression of both cytokines remained significantly elevated compared to that for uninfected controls (IL-4, P < 0.01; IL-13, P < 0.05). Interestingly, for N. brasiliensis-infected SCID mice, transcription of IL-13 was significantly enhanced (P < 0.05) throughout the study (days 2 to 12 p.i.), whereas transcription of IL-4 was repressed during the innate phase of the response to N. brasiliensis infection (Fig. 4B).
Expression of beta interferon was modestly elevated at days 2 and 12 p.i. in WT animals but was downregulated during the innate window in SCID mice (Fig. 4D). Consistent with this lack of expression, genes regulated by type I interferons were not among the upregulated genes in the gene array data from SCID animals (data not shown). The proinflammatory cytokines IL-1, IL-6, and TNF are components of the innate response to pathogen challenge (69). Transcription of IL-1 and IL-6 was rapidly induced as N. brasiliensis larvae entered the lungs of both WT and SCID mice, but only IL-6 expression remained significantly (P < 0.05) elevated beyond day 4 p.i. (Fig. 4C) in both strains of mice. Expression of the gene encoding TNF in the lungs of WT and SCID animals was similar to uninfected control levels throughout the course of N. brasiliensis infection.
IL-10 and TGF- play important roles in the regulation of the magnitude and the scope of inflammatory responses, including responses in the lungs (76). IL-10 gene expression was elevated during the innate response to N. brasiliensis for both WT and SCID mice, and this upregulation persisted through day 12 p.i. (Fig. 4B). In contrast, TGF- transcription was not increased at any time during N. brasiliensis infection for WT and SCID animals (Fig. 4B).
Expression of genes encoding the chemokines CCL8, CCL9, CCL11, CCL17, CXCL1, and CXCL13 was significantly elevated in both SCID and WT lungs during the innate immune response to N. brasiliensis infection (Fig. 3; Table 1). Only WT animals maintained elevated transcription levels of these chemokines beyond the innate response to N. brasiliensis. It is interesting to note that the lungs of N. brasiliensis-infected animals had no significant infiltration of neutrophils or eosinophils (data not shown) despite significantly elevated transcription of the genes encoding CCL8 and CCL11.
N. brasiliensis induces genes associated with alternatively activated macrophages. Previous studies have demonstrated the presence of AAMs in the context of the strong Th2 cytokine environment produced during nematode infections (37, 53). AAMs have been shown to synthesize a distinctive set of gene products that include the resistin-like secreted protein (FIZZ1), a chitinase-like lectin (YM1), arginase (ARG1), and the mannose receptor C1 (Mrc1) (22, 48, 56, 73). Transcription of the AAM-associated genes ym1, fizz1, and mrc1 was significantly enhanced in the lungs of both WT and SCID animals during the innate responses against N. brasiliensis both as measured by microarray (Fig. 3; Table 1) and confirmed by real time RT-PCR (P < 0.05) (Fig. 4A). The levels of transcription for most AAM-associated genes were comparable in WT and SCID lungs during the first 4 days of infection (Fig. 4A). In the lungs from SCID animals, the transcription levels dropped significantly between days 4 and 12 p.i., while cells in the lungs from N. brasiliensis-infected WT animals maintained or increased transcription levels (Table 1; Fig. 4A). In contrast to the dramatic increase in fizz1 expression, there was no increase in the expression of the related molecule fizz2 (see Table S3 in the supplemental material).
The results of the PCR analyses indicated that the overall level of transcription of arg1 was low in the lungs prior to and after N. brasiliensis infection, but there was an increase in expression during the innate response in both WT and SCID animals (Fig. 4A).
The expression profile of ym1 reported here confirms the observations made by Nair et al. (53) that ym1 was induced as early as 24 h and as late as 15 days post-N. brasiliensis infection. The gene array results demonstrated that the gene encoding YM2, a highly related paralogue of YM1, was also enhanced in both WT and SCID lungs in response to N. brasiliensis infection (Table 1). Because of the degree of sequence identity between ym1 and ym2, the transcription of ym2 was validated by a ym2-specific primer-probe set in real-time RT-PCR (Fig. 4A). This finding is in contrast to a previous report that N. brasiliensis induces only ym1 expression in the lung (53) but is consistent with the coexpression of ym1 and ym2 in the lung after allergen challenge (17, 72). The gene encoding another member of the chitinase family, the murine orthologue of the acidic mammalian chitinase (AMCase) (5), also showed enhanced expression in the lungs of N. brasiliensis-infected animals as early as day 2 p.i. (Table 1). This confirms a previous report of N. brasiliensis-induced expression of murine AMCase in the lungs at day 6 p.i. (53). The functional significance of this coordinated production of three members of the chitinase family of proteins during the innate response to N. brasiliensis infection is not clear, but it suggests that they may play a role in lung homeostasis after acute injury.
N. brasiliensis infection induces alternatively activated alveolar macrophages. The expression profiling results predicted that N. brasiliensis infection induced a dramatic increase in the number of AAMs in the lungs. An immunohistocytochemical approach was utilized to determine the dynamics of AAMs in the lungs of WT and SCID mice. Alveolar macrophages were identified in histological sections by their ability to bind GSL I, a lectin that binds selectively to -galactose residues on the surfaces of alveolar macrophages (61, 66) (Fig. 5A). All GSL I+ alveolar macrophages were also F4/80+ (data not shown). AAMs were identified by their coexpression of YM1 and GSL I (Fig. 5A). All YM1+ cells were also GSL I+. YM1 appeared to be localized predominantly in perinuclear cytoplasmic vesicles (Fig. 5A).
By day 4 p.i., there was a significant increase in the number of GSL I+ alveolar macrophages in both WT and SCID mice, and these elevated levels were maintained through day 12 p.i. (Fig. 5C). From a low number in uninfected animals, there were rapid and significant increases in the YM1+ AAMs in WT and SCID mice at day 2 p.i. (Fig. 5C). As observed for the GSL I+ cells at day 2 p.i., there was a statistically significant increase (P < 0.01) in the number of YM1+ AAMs found in SCID lungs compared to that found in lungs from WT mice. The YM1+ AAMs were uniformly distributed throughout the parenchyma of the lung (Fig. 5B) for both strains of mice. In WT animals, the YM1+ AAM numbers increased further at day 4 p.i. and were maintained at elevated levels through day 12 p.i. In contrast, YM1+ AAM levels in the lungs of SCID mice decreased significantly (P < 0.01) by day 12 p.i. from the peak at day 4 p.i. The drop in AAM numbers in the lungs of SCID mice paralleled the decrease observed in the transcription of AAM-associated genes (Fig. 4A). After infection, >90% of the GSL I+ cells were also YM1+, suggesting that the alveolar macrophages had adapted the AAM phenotype. The differences in the proportions of these alternatively activated alveolar macrophages (AAAM) in WT and SCID animals beyond day 4 p.i. parallel the decrease in ym1 transcription and indicate that adaptive immune responses are responsible for persistence of the AAAM phenotype in the lungs.
DISCUSSION
Nippostrongylus larvae induced an immediate, robust innate immune response in the lungs from both WT and immunodeficient BALB/c mice. The cellular and molecular events that trigger this strong innate immune response are not known. Whereas the receptors and the signaling pathways that trigger innate immune responses against a number of viral, bacterial, fungal, and protozoan pathogens have been identified (4), the receptors that are important for innate recognition of most helminth infections are still not clear. Recent results suggest that glycans and double-stranded RNA from helminth parasites can activate Toll-like receptors (1, 14, 21), but the role this activation plays in shaping the adaptive immune response and in resistance to infection is yet to be defined. The response to N. brasiliensis in the rodent lung offers a model system to test the role of innate immune receptors in responses to parasitic nematodes.
A number of major parasitic helminths of humans, such as hookworms, Ascaris, and Schistosoma, have a transient residency in the lungs as part of their life cycles. Typically this takes place early in the life cycle when the parasite is a relatively small larva and the stay in the lungs is of short duration. Presumably, this obligate developmental phase in the lungs provides an advantage to the parasite, but at this time the nature of this advantage is not clear. It is possible that the larvae receive host-derived developmental signals in the lung environment. It is also possible that the acute inflammation induced in the pulmonary environment by the parasite modifies or "conditions" the immunological environment in a way that confers a survival advantage for the subsequent developmental stages. The parasites could be exploiting the inherent mechanisms in place in the lung to regulate inflammation (25, 35). The regulatory mechanisms initiated in the lung might modulate the magnitude and the quality of subsequent immune responses against the parasite. In support of this idea are reports that a helminth infection modifies the immune responses to a number of pathogen and nonpathogen challenges (34, 43, 45, 76). Indeed, pathogen-induced counterregulatory mechanisms in the lungs are a basic tenet of the hygiene hypothesis (75, 78).
To our knowledge this is the first report on the histopathology of the lung phase of N. brasiliensis infection in mice. In WT BALB/c mice, there was a remarkably rapid resolution of mechanical damage and inflammation caused by N. brasiliensis larvae migrating through the pulmonary environment (Fig. 1). Within hours of the larvae exiting the lungs, the hemorrhage and inflammatory infiltrate were largely resolved, and by day 8 p.i., the histological appearance of the parenchyma of the lung and the large airways was nearly indistinguishable from that for uninfected controls (see inserts in Fig. 1). This rapid resolution took place in the context of cytokines IL-4 and IL-13, which have been demonstrated to mediate inflammation, epithelial cell hyperplasia, and mucus production in the lung (reviewed in reference 74). In contrast, the larva-induced inflammation in the lungs of SCID animals persisted through day 8 p.i., with significant levels of cellular infiltrate evident throughout the lung (Fig. 1H). The mechanistic basis for this difference in the ability to resolve pulmonary inflammation between WT and SCID animals is not clear but presumably reflects a defect in lymphocyte-mediated homeostatic mechanisms in the SCID animals.
Comparing the expression profiles from the lungs of infected WT and SCID mice provided an opportunity to study the complexities of the immediate innate immune response to N. brasiliensis larvae, to characterize the transition between the innate and adaptive immune mechanisms, and to infer the role of T cells in sustaining responses initiated by the innate immune system. Although there were differences, the dynamics and magnitude of the transcriptional and cellular responses in the lungs from WT and SCID mice associated with innate immune responses were similar during the first 4 days of infection. In the SCID mice, there was a rapid decay in many of the N. brasiliensis-induced cellular and transcriptional responses after day 4 p.i. (Table 1; Fig. 5C); however, transcription of a few genes, such as those encoding IL-10 and IL-13 (Fig. 4), as well as the number of alveolar macrophages (Fig. 5B), remained elevated. In contrast, a significant percentage of the transcriptional and cellular responses initiated during the innate phase were maintained or enhanced in the lungs of WT mice. The results underscore a complex regulation of inflammation in the lungs, with certain aspects under control of T cells while other facets are regulated in a T cell-independent fashion.
One of the most marked transcriptional changes observed in both WT and SCID animals was the immediate upregulation of the genes associated with AAM: ym1, ym2, fizz1, and arg1. Resident macrophages in different tissues adapt to their local environments based on interactions with secreted factors, surface signals from neighboring cells, and the nature of the extracellular matrix. Once established in the tissues, macrophages can be activated in a variety of ways. Classical macrophage activation occurs through stimulation from lymphocyte-derived IFN- and is key for the development of effective cell-mediated immune responses against intracellular pathogens (12). An alternative pathway of macrophage activation has also been defined that is mediated by IL-4 and/or IL-13 (22). IL-4/IL-13 activation of macrophages upregulates the mannose receptor (16), class II major histocompatibility complex, and CD80/CD86 (13) on their surfaces. It is generally thought that AAMs function in debris scavenging, tissue remodeling, wound healing, and the promotion of Th2 immune responses (22, 49, 63). It has also been demonstrated that AAMs inhibit T-cell proliferation (37, 41). The mechanisms through which AAMs carry out these functions are not clear. However, AAMs secrete a characteristic set of molecules, including YM1 (38), FIZZ1, and arginase (51), that are likely to contribute to the function of these cells.
YM1 is a member of a family of proteins that share sequence similarity to chitinases from lower organisms (29). Transcripts encoding YM1 can represent over 10% of the total nematode-induced AAM mRNA (38). YM1 has the ability to bind to chitin and related glycan structures (8), but its apparent lack of demonstrable chitinase activity makes its role as an effector molecule unclear. YM1 might play a defensive role by binding chitin-containing pathogens, such as fungi. for subsequent recognition by immune cells. Through its ability to bind to the extracellular matrix, YM1 might also mediate cellular trafficking that promotes wound healing and matrix repair—one of the proposed functions of AAMs (24, 59).
The functional significance of FIZZ1 is also yet to be defined. FIZZ1 (found in inflammatory zone I; also called resistin-like molecule ) was initially described as a product found in the bronchoalveolar lavage fluid of mice challenged with an allergen that was capable of inhibiting nerve growth factor in vitro (26). Subsequently it has been demonstrated that FIZZ1 is produced by macrophages in response to stimulation by IL-4 (38, 53, 57). FIZZ1, like YM1, has been implicated in regulating the deposition of extracellular matrix in the lungs (36), thus linking these two molecules to a role in pulmonary repair and remodeling. Also consistent with the previous report, we observed only a minor increase in the transcription of fizz2 in the lungs after N. brasiliensis infection (see Table S2 in the supplemental material). fizz2 appears to be preferentially expressed in the intestine (53, 64).
The balance between inducible nitric oxide synthase (iNOS) and arginase 1 is a central feature in the development of functionally distinct macrophage populations. iNOS and arginase 1 compete for L-arginine to catalyze the production of NO/L-citrulline or urea/L-ornithine, respectively. IL-4/IL-13 stimulate the production of arginase 1 and inhibit iNOS, while IFN- inhibits arginase and promotes iNOS production (24, 50). In the Th2 environment induced by N. brasiliensis infection, there was no increase in the expression iNOS (data not shown). Like YM1 and FIZZ1, arginase 1 is associated with repair and remodeling of tissues, specifically with collagen deposition and fibrogenesis (51).
Previous studies have shown that the induction of AAM-associated genes is dependent on IL-4 or IL-13 (22, 41, 73). The timing of the response in WT mice and the presence of the response in SCID mice indicate that T cells were not the source of these Th2 cytokines. The most likely source for this rapid production of IL-4 and IL-13 in the lungs is granulocytic cells—eosinophils, mast cells, or basophils (20, 62). Eosinophils, mast cells, and basophils have the capability of uncoupling transcription and translation of IL-4 and IL-13. These cells have been shown to constitutively transcribe IL-4 and IL-13 to form a reservoir of transcripts that can be mobilized for immediate cytokine production upon stimulation (20). Once activated, these cells can then continue to transcribe and produce IL-4 and IL-13. This ability to maintain cytokine synthesis after activation might explain the sustained production of IL-13 in the lungs of SCID animals (Fig. 4B). The significant increase in the transcription of IL-4 and IL-13 in WT lungs after day 4 p.i. is presumably due to T-cell-mediated adaptive responses.
The dynamics of IL-4 and IL-13 transcription differed significantly in WT and SCID lungs after N. brasiliensis infection (Fig. 4B). In the lungs from SCID animals, there was an immediate and selective upregulation of IL-13 transcription after N. brasiliensis infection that was not seen in WT animals. The cellular and molecular basis for this differential regulation between IL-13 and IL-4 is not clear (Fig. 4B), but it is likely to reflect fundamental differences between WT and SCID animals in the numbers and activation status of the cells in the lung that respond to innate signals. The cellular infiltrate induced by N. brasiliensis at days 2 and 4 p.i., measured by bronchial alveolar lavage, was dominated by macrophages, with only minor increases in eosinophil and neutrophil numbers (data not shown). The lack of significant changes in the transcription levels of eosinophil- and mast cell-associated genes in the expression profiling of N. brasiliensis-infected animals (data not shown) also indicated there was no major influx of these cells into the lungs. Recently, it has been demonstrated that basophils are a major source of IL-4 and that the number of IL-4-producing basophils in the lungs is increased 50-fold by N. brasiliensis infection (47). This increase in basophil levels is T cell dependent. It is possible that the lack of IL-4 production in the lungs of SCID mice reflects the defect in T-cell-mediated recruitment of basophils to the lungs. The major immediate cellular source of IL-13 in SCID lungs is yet to be determined.
AAMs have been shown to be induced outside the lung by other nematode infections (29, 37, 41, 53), as well as by digenetic trematodes (15, 18, 23), cestodes (7, 58), and protozoa (27, 57). The IL-4-dependent AAMs induced in the peritoneal or pleural cavities by the filarial nematodes Brugia malayi and Litomosoides sigmodontis, respectively, exerted a profound, contact-dependent antiproliferative effect on a range of different cell types, including antigen-specific T cells (37, 41, 67). Consistent with these previous observations, the AAAMs from N. brasiliensis-infected animals expressed class II major histocompatibility complex and readily took up large amounts of antigen but suppressed antigen-specific T-cell proliferation (data not shown). The mechanism through which AAMs suppress cell proliferation has not been defined. AAAMs could reflect a homeostatic mechanism in the lungs that functions to suppress inflammation after certain antigenic challenges, especially those that induce strong Th2 responses.
One of the complicating factors in assessing the innate response to N. brasiliensis is the inability to discriminate between the responses to parasite somatic and secreted antigens and the response induced by the focal mechanical damage caused by larval migration through the lungs. While it is not possible to evaluate the relative contributions of these two facets of larval invasion with the experimental design presented here, a survey of reports on the immediate transcriptional responses associated with chemical- or ventilator-induced acute lung injury show significant differences in the gene expression profiles from that induced by N. brasiliensis infection (39, 44, 46). While there are expected overlaps in the expression of genes involved in protection against oxidant injury, cell proliferation, and extracellular matrix repair, there is a notable lack of expression of genes associated with a Th2 response and AAM activation in the lungs of mice undergoing acute lung injury. Further experiments are required to determine the relative roles of parasite antigens and mechanical disruption to the signature expression profile induced by N. brasiliensis infection.
Immunohistochemical analysis of lungs from uninfected animals showed that YM1 was constitutively produced at modest but detectible levels by a subset of the GSL I+ alveolar macrophages (also F4/80+), which is consistent with a previous report of YM1 production in the lungs of adult mice (29). This constitutive production of YM1 in the lungs was supported by the intensity values of the transcriptional data (see Table S4 in the supplemental material). In addition, genes encoding other AAM-associated molecules, such as FIZZ1, MRC1, and AMCase but not Arg1, were also expressed in noninflamed lungs (see Table S4 in the supplemental material). These observations suggest that 15% of the cells typically referred to as alveolar macrophages are AAAMs (Fig. 5C). It is possible that the proportion might even be higher. One interpretation of the rapid conversion of the lungs from an environment that is populated with macrophages which are predominantly GSL I+/YM1– to an environment that is dominated by GSL I+/YM1+ cells is that the alveolar macrophages were directly converted to the AAAM phenotype as part of the innate response to N. brasiliensis. It is interesting to speculate that alveolar macrophages are incipient AAAMs that require innate or adaptive signals to fully mature. Like AAM, alveolar macrophages are commonly characterized as anti-inflammatory and have been shown to participate in tissue repair and remodeling (55). It is also possible that there is a rapid efflux and influx of cells into the lungs and that a majority of the cells that enter the lungs are AAM. Additional work is needed to define the trafficking of macrophages into and out of the lungs during inflammation and how the dynamics of trafficking influences macrophage populations.
.
ACKNOWLEDGMENTS
This work was supported by grants from NIH NHLBI (U01 HL66623) and NIH training grant T32AI007417.
We thank Joseph Urban (USDA, Beltsville, MD) for providing Nippostrongylus brasiliensis, Anne Jedlicka (Johns Hopkins Malaria Research Institute Gene Array Core Facility) for assistance in the gene array experiments, and Brian Schofield (Johns Hopkins University) for his assistance with immunohistocytochemistry.
FOOTNOTES
Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205. Phone: (410) 955-3430. Fax: (410) 955-0105. E-mail: ascott@jhsph.edu.
Supplemental material for this article may be found at http://iai.asm.org/.
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ABSTRACT
While it is well established that infection with the rodent hookworm Nippostrongylus brasiliensis induces a strongly polarized Th2 immune response, little is known about the innate host-parasite interactions that lead to the development of this robust Th2 immunity. We exploited the transient pulmonary phase of N. brasiliensis development to study the innate immune responses induced by this helminth parasite in wild-type (WT) and severe-combined immune deficient (SCID) BALB/c mice. Histological analysis demonstrated that the cellular infiltrates caused by N. brasiliensis transit through the lungs were quickly resolved in WT mice but not in SCID mice. Microarray-based gene expression analysis demonstrated that there was a rapid induction of genes encoding molecules that participate in innate immunity and in repair/remodeling during days 2 to 4 postinfection in the lungs of WT and SCID mice. Of particular note was the rapid upregulation in both WT and SCID mice of the genes encoding YM1, FIZZ1, and Arg1, indicating a role for alternatively activated macrophages (AAMs) in pulmonary innate immunity. Immunohistochemistry revealed that nearly all alveolar macrophages became YM1-producing AAMs as early as day 2 postinfection. While the innate responses induced during the lung phase of N. brasiliensis infection were similar in complexity and magnitude in WT and SCID mice, only mice with functional T cells were capable of maintaining elevated levels of gene expression beyond the innate window of reactivity. The induction of alternatively activated alveolar macrophages could be important for dampening the level of inflammation in the lungs and contribute to the long-term decrease in pulmonary inflammation that has been associated with helminth infections.
INTRODUCTION
The cells and molecules that comprise the innate immune responses are the first line of defense against invading pathogenic organisms. In addition, innate immune mechanisms are important for the elimination of the myriad of nonpathogenic substances to which we are continuously exposed. In those circumstances in which the effector mechanisms of the innate response are not sufficient to fully eliminate a pathogen challenge, the same cells and molecules function to efficiently activate the antigen-specific adaptive arm of the immune response. In recent years, it has become clear that different modes of activation of innate immunity have a profound influence on the magnitude and the nature of subsequent adaptive responses (9, 32, 69).
Parasitic nematodes are among the strongest natural inducers of polarized Th2 immune responses (2, 40, 60). Infection of mice with the intestinal nematode parasite Nippostrongylus brasiliensis has been used extensively to study the regulation of immunoglobulin E synthesis (31) and Th2 immunity in general (19, 33). Despite the longstanding use of N. brasiliensis as a model, little is known about the innate immune responses that precede the induction of a highly polarized adaptive Th2 response to N. brasiliensis challenge. Indeed, there are only limited data on the dynamic interplay between helminth antigens and the receptors of the innate immune system of mammals (1, 3, 21, 68). Given the exceedingly high prevalence of helminth infections in human and animal populations (6, 11) and the recent appreciation that helminth infections influence subsequent responses to other pathogens (65), environmental antigens (76), and vaccines (10, 52), defining the innate-mediated responses induced by helminth parasites will provide a basis for understanding the downstream cellular and molecular events that lead to highly regulated immune responses.
In addition to being a tested model for the study of Th2 immune responses, N. brasiliensis infection in mice serves as a model for human hookworm infections with Necator americanus and Ancylostoma duodenale. Human and rodent hookworms, as well as a number of other helminth species of public health importance, migrate through the lungs of the host during larval development. This obligate pulmonary phase of the life cycle typically occurs within a few hours after infection and lasts only about a day before the worms migrate to the intestine, where they develop into adults. The significance of this transient migration through the lungs to the physiology of the parasite is not clear. However, one of the consequences of this brief exposure on the host is a substantive, prolonged alteration of the immunological status of the lung. For example, the level of allergen-induced pulmonary inflammation was significantly less in mice that were previously infected with N. brasiliensis than in noninfected controls (42). The results derived from the N. brasiliensis-mouse model are consistent with the inverse correlations made in human populations between helminth infection and the prevalence of allergic asthma (43, 79).
To gain insight into the mechanisms underlying parasite-induced alterations in immunological responsiveness, we examined the lung phase of N. brasiliensis development in wild-type (WT) and severe-combined immune deficient (SCID) mice. SCID mice were employed to study innate responses in an environment devoid of the major cellular mechanism necessary to mount an adaptive immune response. N. brasiliensis induced a rapid and robust innate immune response in the lungs of both WT and SCID mice. Expression profiling showed that both strains of mice transcribed a similar subset of genes during the innate response to N. brasiliensis infection. Of particular note was the strong expression of genes associated with alternatively activated macrophages: ym1, fizz1, and arg1. The presence of alternatively activated macrophages (AAMs) in the lungs of both WT and SCID animals was confirmed by immunohistocytochemistry. Although the initial responses to N. brasiliensis were similar in complexity and magnitude in the lungs of WT and SCID mice, only WT mice were capable of maintaining elevated levels of gene expression beyond the innate window of reactivity. These results provide novel insights into the cellular and molecular basis for infection-induced modulation of pulmonary immune reactivity.
MATERIALS AND METHODS
Animals and infection. Six- to eight-week-old male WT and SCID mice on a BALB/c background were obtained from the National Cancer Institute at Frederick, Md. Mice were housed in barrier filter-top cages, given food and water ad libitum, and kept on a 12-h light/dark cycle. Infectious third-stage N. brasiliensis larvae were harvested from a fecal culture via a Baermann apparatus, washed in phosphate-buffered saline, and counted. Mice were infected subcutaneously with 500 larvae. All experimental procedures described in this paper were performed under the approval of the Johns Hopkins University Animal Care and Use Committee in accordance with the guidelines set out by the National Research Council's Guide for the Care and Use of Laboratory Animals.
Histology. (i) Lung inflations. Animals were anesthetized with 300 mg/kg of body weight of Avertin (2,2,2-tribromoethanol) administered intraperitoneally. Pericardium and trachea were exposed by dissection. A lateral incision was made through the trachea, and a 19-gauge flat-tipped needle was inserted and tied off. Inflation of the lungs was performed slowly over 2 min with 0.8 ml of zinc-buffered formalin fixative (Z-fix) (Anatech Ltd., Battle Creek, MI). The trachea was then tied off, and the lungs were removed from the pericardial cavity and incubated in a 40-fold volume of Z-fix.
(ii) Histological preparation. Inflated and fixed lungs were embedded in paraffin, and sagittal 10-μm sections were obtained from four different levels of the lung. Sections were stained with hematoxylin and eosin (H&E) or with periodic acid Schiff plus hematoxylin counterstain.
(iii) Immunohistochemistry. Sections were stained for alveolar macrophages using biotinylated Griffonia (Bandeiraea) Simplicifolia lectin 1 (GSL I) (10 μg/ml; Vector Laboratories, Burlingame, CA) followed by incubation with alkaline phosphatase-conjugated streptavidin (1:200) (Vector Laboratories) and Liquid Permanent Red chromogen (DAKO USA, Carpinteria, CA). YM1 staining was performed using affinity-purified goat antimouse YM1 (1 μg/ml; R&D Systems, Minneapolis, MN). Anti-YM1 was detected using a biotinylated rabbit antigoat immunoglobulin G (Vector Laboratories), peroxidase streptavidin and 3,3'-diaminobenzidine (Vector Laboratories). For dual-fluorescence staining of macrophages, YM1 was resolved with Fluorescein/Avidin DCS (Vector Laboratories) and, after an avidin/biotin blocking step (Vector Laboratories), GSL I binding was detected with Texas Red/Avidin DCS. Stained sections were mounted in Vectashield containing 4'-6'-diamidino-2-phenylindole (Vector Laboratories).
(iv) Enumeration. The values from three WT and three SCID animals at 0, 2, 4, 8, and 12 days postinfection were used to evaluate the changes in alveolar macrophage and AAM numbers. The GSL I-positive and YM1-positive cells were counted in each of six contiguous lung histological fields (magnification, x10; 974 mm2/field), and the mean cell number per field was calculated. Statistical significance was determined using a two-tailed paired Student t test.
(v) Light microscopy and imaging. Lung sections were examined using a Nikon Eclipse E800 light microscope (Nikon, Inc., Melville, NY), and images were acquired using a SPOT RT charge-coupled-device imager and software (Diagnostic Instruments, Inc., Sterling Heights, MI).
Gene expression analysis. (i) Total RNA extraction. Lungs were harvested, flash frozen in liquid nitrogen, and stored at –80°C. Lungs were homogenized in 2 ml Trizol (Invitrogen, Carlsbad, CA), and 1 ml of homogenate was processed for RNA isolation according to the manufacturer's (Invitrogen) protocol with minor modifications including the use of glycogen at 10 μg/ml as a carrier for an overnight isopropanol precipitation and increasing centrifugation times to 15 min. RNA pellets were resuspended in nuclease-free water. RNA quality was determined by RNA Nano LabChip analysis on a Bioanalyzer 2100 (Agilent, Palo Alto, CA). An RNeasy total RNA cleanup protocol (QIAGEN) was performed, followed by spectrophotometric assessment of the RNA concentration. The lungs from three animals were independently processed for each treatment group and each time point.
(ii) Affymetrix GeneChip protocols. Processing of templates and hybridization for the 430 2.0 array GeneChip (Affymetrix, Inc, Santa Clara, CA) was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision 3, as previously described (30). Following hybridization, the GeneChips were washed and stained in an automated fluidics station (Affymetrix FS450) and then assessed using the GCS3000 laser scanner (Affymetrix) at an emission wavelength of 570 nm at a 2.5-μm resolution. The intensity of hybridization for each probe pair was computed by GCOS 1.2 software (Affymetrix). (For more-detailed methods, please refer to the website of the Malaria Research Institute Gene Array Core Facility at the Johns Hopkins Bloomberg School of Public Health [http://jhmmi.jhsph.edu]).
(iii) Data analysis. Affymetrix CEL file data were preprocessed for use with the probe level GeneChip Robust Multi-Array analysis (77) option in GeneSpring 7 Software (Agilent Technologies). Initial filtering by probe intensity for raw levels above 150 in at least 2 out of 12 conditions resulted in a list of 12,036 genes that were then used as the basis for selecting differentially regulated genes. Raw intensity values ranged from 150 to 46,037.
Real-time RT PCR. From each treatment group and time point, 1 μg of lung RNA was reverse transcribed using the SuperScript first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using an oligo(dT) primer. Resultant cDNAs were amplified for real-time detection with fluorogenic-labeled probes in assays specific for each target gene. Quantitative real-time RT-PCR was performed using the Applied Biosystems 7500 real-time PCR system, TaqMan gene expression assays-on-demand, and TaqMan Universal master mix (Applied Biosystems, Foster City, CA). The following assays (Applied Biosystems product numbers) were used: YM1 (Mm00657889_mH), YM2 (Mm00840870_m1), FIZZ1 (Mm00445109_m1), ARG1 (Mm00475988_m1), interleukin 4 (IL-4) (Mm00445259_m1), IL-13 (Mm00434204_m1), tumor necrosis factor (TNF) (Mm00443258_m1), IL-1 (Mm00434228_m1), IL-10 (Mm00439616_m1), transforming growth factor beta (TGF-) (Mm00441724_m1), IL-6 (Mm00446190_m1), beta-1 interferon (Mm00439546_s1), gamma interferon (IFN-) (Mm00801778_m1), and alpha-2 interferon (Mm00833961_s1). Reactions were performed using 1 μl of cDNA in a 25-μl sample volume and the following thermal cycler profile: 10 min of denaturation at 95°C, 50 cycles of 1-min extension at 60°C and then 15 s of denaturation at 95°C. Analysis was performed using the 7500 system SDS software package (Applied Biosystems).
Microarray data accession number. The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through Gene Expression Omnibus series accession number GSE3414.
RESULTS
Nippostrongylus in the lung. After subcutaneous infection, N. brasiliensis infective-stage larvae entered the general circulation and were carried to the lungs. Between 24 and 48 h postinfection (p.i.), the larvae penetrated alveolar capillaries and transiently took up residence in the alveolar space (Fig. 1I). In both WT and SCID BALB/c mice, focal damage to the alveolar capillaries resulted in hemorrhage into the air space (Fig. 1C and D). By day 4 p.i., the larvae had exited the lungs and entered the small intestine, where they molted to adults, and the females released eggs for 3 to 5 days prior to being expelled from the gut via an immune-mediated mechanism (data not shown) (71). N. brasiliensis was not expelled from the intestine of the SCID mice, since CD4+ T cells are required for resolution of infection (70, 71).
At day 4 p.i. (24 to 36 h after the larvae have exited the lungs), the hemorrhage was largely resolved in WT mice and there was evidence of a modest influx of mononuclear cells (Fig. 1E). In contrast, hemorrhage in the lungs from SCID animals persisted, and the mononuclear cell infiltrate was elevated compared to WT levels at days 4 and 8 p.i. (Fig. 1E through H). In addition to the changes in the parenchyma of the lung, the presence of N. brasiliensis larvae induced epithelial cell and goblet cell hyperplasia (Fig. 1C to H, inserts, and J). The hyperplasia was largely resolved in WT lungs by day 8 p.i. (Fig. 1G) but persisted in the SCID mice (Fig. 1H).
N. brasiliensis larvae induce similar innate responses in the lungs of SCID and WT mice. A gene expression profiling approach was used to study the innate immune response in the lungs during the pulmonary phase of N. brasiliensis infection in WT and SCID BALB/c mice. Lungs from infected animals were removed at days 0, 2, 3, 4, 8, and 12 p.i. and processed for gene expression analysis. Of special interest were genes that were significantly upregulated between days 0 and 4 of infection, corresponding to the period where innate immune responses are dominant. A comparison of the expression profiles in uninfected WT and SCID BALB/c mice showed that just under 7% of the 11,500 genes expressed in the lungs were differentially transcribed in the two strains (data not shown). WT mice differentially transcribed genes associated with adaptive immunity (B- and T-cell-related genes), which were predictably absent in SCID mice. Based on these similarities in expression profiles, gene expression data from N. brasiliensis-infected WT and SCID mice were normalized to the expression levels in strain-matched uninfected lungs.
On day 2 of infection, the newly arrived N. brasiliensis larvae induced at least a twofold increase in the transcription of 148 and 112 genes in the lungs of WT and SCID mice, respectively, compared to the expression profiles of the strain-matched noninfected controls (Fig. 2A). Approximately 35% of the N. brasiliensis-induced genes were common to both WT and SCID lungs at day 2 (Fig. 2A). On days 3 and 4 of infection, an additional 69 and 115 genes were upregulated in WT and SCID animals, respectively, of which 22% were common to both strains. Of the total of 309 different genes whose transcription was significantly augmented by N. brasiliensis at days 2 to 4 of infection in both WT and SCID mice, a direct role in innate immunity as defined by GO slims annotation could be ascribed to approximately one-fourth (76 genes) (see Table S1 in the supplemental material), and 58% (44 genes) of these were induced in both WT and SCID mice. Among the genes upregulated in the lungs of WT (217 genes) and SCID (227 genes) animals on days 2, 3, and 4 of infection (Fig. 2B and C), 72 (33%) and 54 (24%), respectively, had significantly enhanced transcription at all three time points. Over the course of infection, the percentage of genes that concomitantly increased in WT and SCID lungs peaked at 44% at day 4 p.i. and dropped to under 5% at day 12 p.i. (Fig. 2A). By day 12 p.i., only 14 genes were significantly upregulated in the lungs of SCID mice, whereas WT mice maintained an elevated transcription of 78 genes (Fig. 2A; see Table S2 in the supplemental material). Thus, while both WT and SCID mice are capable of rapidly initiating an innate immune response to the migration of N. brasiliensis larvae in the lungs, only WT BALB/c animals are capable of sustaining the response.
With a focus on genes encoding proteins with the potential to participate in innate immune responses, 70 genes had significantly enhanced transcription for at least one time point during the first 4 days of infection of WT or SCID lungs (Fig. 3; Table 1; see Table S3 in the supplemental material). In general, the N. brasiliensis-induced genes showed comparable trends of upregulation in WT and SCID mice (Fig. 3 and 4; Table 1) during the innate window of immune response. In the lungs of SCID mice, many of these genes dropped back to baseline levels after day 4 p.i. (Fig. 3 and 4; Table 1). In contrast, the transcription of most of these genes was maintained or increased in WT mice through day 12 p.i.
Genes encoding proteins associated with allergen-induced Th2 responses in the lungs, such as trefoil factor 2 (TFF-2) (54), small proline-rich protein 2 (SPRR-2) (81), and Gob-5 (80), were also found to be upregulated in the lungs of N. brasiliensis-infected WT and SCID mice (Table 1). Of particular note, transcription of gob-5 was significantly elevated through day 12 p.i. in the lungs of WT animals, whereas gob-5 expression was robust but limited to the innate window in the lungs of SCID mice. Expression of the genes encoding the IL-13- and STAT6-dependent SPRR-2a (81) and TTF-2 (54) were largely restricted to the lungs from WT animals at 8 to 12 days p.i., suggesting that these genes might be under control of adaptive immune mechanisms.
The hypothesis that helminths have evolved mechanisms of evading host defense by suppressing inflammatory responses has been discussed in previous studies (43, 79). The results of our array experiments suggest that downregulation is not a major mechanism during the innate immune response, and helminth-mediated immunomodulation is confined to adaptive immunity. Of the 277 genes that were downregulated more than twofold at any given time point in either WT or SCID mice, less than 7% had an immune-associated function (data not shown).
N. brasiliensis larvae induce repair and remodeling in the lung. The innate inflammatory response induced by larval migration initiated transcription of genes encoding molecules key to lung repair and remodeling, including elastin, procollagen, matrix metalloprotease (MMP12), and cysteine protease (cathepsin K), and extracellular matrix proteins (fibronectin and tenascin C) (Fig. 3; Table 1). In addition, the transcription of the tissue inhibitor of metalloprotease 1 gene was also significantly upregulated, which presumably played a role in limiting the action of MMP12 in the lungs (28). While expression of these genes was induced to comparable levels in the lungs from both WT and SCID mice during the first 4 days of infection, only WT animals were capable of maintaining an elevated level of expression. The results indicate that lymphocytes play a key role in the regulation of a sustained repair process in the lung.
Cytokines and chemokines. IL-4 and IL-13 are key cytokines in the induction and promotion of Th2 immune responses (33). During the innate immune response to N. brasiliensis in the lungs of WT mice, the transcription of the genes encoding IL-4 and IL-13 was variable until day 4 p.i. (Fig. 4B). Between days 4 and 12 p.i., the expression of both cytokines remained significantly elevated compared to that for uninfected controls (IL-4, P < 0.01; IL-13, P < 0.05). Interestingly, for N. brasiliensis-infected SCID mice, transcription of IL-13 was significantly enhanced (P < 0.05) throughout the study (days 2 to 12 p.i.), whereas transcription of IL-4 was repressed during the innate phase of the response to N. brasiliensis infection (Fig. 4B).
Expression of beta interferon was modestly elevated at days 2 and 12 p.i. in WT animals but was downregulated during the innate window in SCID mice (Fig. 4D). Consistent with this lack of expression, genes regulated by type I interferons were not among the upregulated genes in the gene array data from SCID animals (data not shown). The proinflammatory cytokines IL-1, IL-6, and TNF are components of the innate response to pathogen challenge (69). Transcription of IL-1 and IL-6 was rapidly induced as N. brasiliensis larvae entered the lungs of both WT and SCID mice, but only IL-6 expression remained significantly (P < 0.05) elevated beyond day 4 p.i. (Fig. 4C) in both strains of mice. Expression of the gene encoding TNF in the lungs of WT and SCID animals was similar to uninfected control levels throughout the course of N. brasiliensis infection.
IL-10 and TGF- play important roles in the regulation of the magnitude and the scope of inflammatory responses, including responses in the lungs (76). IL-10 gene expression was elevated during the innate response to N. brasiliensis for both WT and SCID mice, and this upregulation persisted through day 12 p.i. (Fig. 4B). In contrast, TGF- transcription was not increased at any time during N. brasiliensis infection for WT and SCID animals (Fig. 4B).
Expression of genes encoding the chemokines CCL8, CCL9, CCL11, CCL17, CXCL1, and CXCL13 was significantly elevated in both SCID and WT lungs during the innate immune response to N. brasiliensis infection (Fig. 3; Table 1). Only WT animals maintained elevated transcription levels of these chemokines beyond the innate response to N. brasiliensis. It is interesting to note that the lungs of N. brasiliensis-infected animals had no significant infiltration of neutrophils or eosinophils (data not shown) despite significantly elevated transcription of the genes encoding CCL8 and CCL11.
N. brasiliensis induces genes associated with alternatively activated macrophages. Previous studies have demonstrated the presence of AAMs in the context of the strong Th2 cytokine environment produced during nematode infections (37, 53). AAMs have been shown to synthesize a distinctive set of gene products that include the resistin-like secreted protein (FIZZ1), a chitinase-like lectin (YM1), arginase (ARG1), and the mannose receptor C1 (Mrc1) (22, 48, 56, 73). Transcription of the AAM-associated genes ym1, fizz1, and mrc1 was significantly enhanced in the lungs of both WT and SCID animals during the innate responses against N. brasiliensis both as measured by microarray (Fig. 3; Table 1) and confirmed by real time RT-PCR (P < 0.05) (Fig. 4A). The levels of transcription for most AAM-associated genes were comparable in WT and SCID lungs during the first 4 days of infection (Fig. 4A). In the lungs from SCID animals, the transcription levels dropped significantly between days 4 and 12 p.i., while cells in the lungs from N. brasiliensis-infected WT animals maintained or increased transcription levels (Table 1; Fig. 4A). In contrast to the dramatic increase in fizz1 expression, there was no increase in the expression of the related molecule fizz2 (see Table S3 in the supplemental material).
The results of the PCR analyses indicated that the overall level of transcription of arg1 was low in the lungs prior to and after N. brasiliensis infection, but there was an increase in expression during the innate response in both WT and SCID animals (Fig. 4A).
The expression profile of ym1 reported here confirms the observations made by Nair et al. (53) that ym1 was induced as early as 24 h and as late as 15 days post-N. brasiliensis infection. The gene array results demonstrated that the gene encoding YM2, a highly related paralogue of YM1, was also enhanced in both WT and SCID lungs in response to N. brasiliensis infection (Table 1). Because of the degree of sequence identity between ym1 and ym2, the transcription of ym2 was validated by a ym2-specific primer-probe set in real-time RT-PCR (Fig. 4A). This finding is in contrast to a previous report that N. brasiliensis induces only ym1 expression in the lung (53) but is consistent with the coexpression of ym1 and ym2 in the lung after allergen challenge (17, 72). The gene encoding another member of the chitinase family, the murine orthologue of the acidic mammalian chitinase (AMCase) (5), also showed enhanced expression in the lungs of N. brasiliensis-infected animals as early as day 2 p.i. (Table 1). This confirms a previous report of N. brasiliensis-induced expression of murine AMCase in the lungs at day 6 p.i. (53). The functional significance of this coordinated production of three members of the chitinase family of proteins during the innate response to N. brasiliensis infection is not clear, but it suggests that they may play a role in lung homeostasis after acute injury.
N. brasiliensis infection induces alternatively activated alveolar macrophages. The expression profiling results predicted that N. brasiliensis infection induced a dramatic increase in the number of AAMs in the lungs. An immunohistocytochemical approach was utilized to determine the dynamics of AAMs in the lungs of WT and SCID mice. Alveolar macrophages were identified in histological sections by their ability to bind GSL I, a lectin that binds selectively to -galactose residues on the surfaces of alveolar macrophages (61, 66) (Fig. 5A). All GSL I+ alveolar macrophages were also F4/80+ (data not shown). AAMs were identified by their coexpression of YM1 and GSL I (Fig. 5A). All YM1+ cells were also GSL I+. YM1 appeared to be localized predominantly in perinuclear cytoplasmic vesicles (Fig. 5A).
By day 4 p.i., there was a significant increase in the number of GSL I+ alveolar macrophages in both WT and SCID mice, and these elevated levels were maintained through day 12 p.i. (Fig. 5C). From a low number in uninfected animals, there were rapid and significant increases in the YM1+ AAMs in WT and SCID mice at day 2 p.i. (Fig. 5C). As observed for the GSL I+ cells at day 2 p.i., there was a statistically significant increase (P < 0.01) in the number of YM1+ AAMs found in SCID lungs compared to that found in lungs from WT mice. The YM1+ AAMs were uniformly distributed throughout the parenchyma of the lung (Fig. 5B) for both strains of mice. In WT animals, the YM1+ AAM numbers increased further at day 4 p.i. and were maintained at elevated levels through day 12 p.i. In contrast, YM1+ AAM levels in the lungs of SCID mice decreased significantly (P < 0.01) by day 12 p.i. from the peak at day 4 p.i. The drop in AAM numbers in the lungs of SCID mice paralleled the decrease observed in the transcription of AAM-associated genes (Fig. 4A). After infection, >90% of the GSL I+ cells were also YM1+, suggesting that the alveolar macrophages had adapted the AAM phenotype. The differences in the proportions of these alternatively activated alveolar macrophages (AAAM) in WT and SCID animals beyond day 4 p.i. parallel the decrease in ym1 transcription and indicate that adaptive immune responses are responsible for persistence of the AAAM phenotype in the lungs.
DISCUSSION
Nippostrongylus larvae induced an immediate, robust innate immune response in the lungs from both WT and immunodeficient BALB/c mice. The cellular and molecular events that trigger this strong innate immune response are not known. Whereas the receptors and the signaling pathways that trigger innate immune responses against a number of viral, bacterial, fungal, and protozoan pathogens have been identified (4), the receptors that are important for innate recognition of most helminth infections are still not clear. Recent results suggest that glycans and double-stranded RNA from helminth parasites can activate Toll-like receptors (1, 14, 21), but the role this activation plays in shaping the adaptive immune response and in resistance to infection is yet to be defined. The response to N. brasiliensis in the rodent lung offers a model system to test the role of innate immune receptors in responses to parasitic nematodes.
A number of major parasitic helminths of humans, such as hookworms, Ascaris, and Schistosoma, have a transient residency in the lungs as part of their life cycles. Typically this takes place early in the life cycle when the parasite is a relatively small larva and the stay in the lungs is of short duration. Presumably, this obligate developmental phase in the lungs provides an advantage to the parasite, but at this time the nature of this advantage is not clear. It is possible that the larvae receive host-derived developmental signals in the lung environment. It is also possible that the acute inflammation induced in the pulmonary environment by the parasite modifies or "conditions" the immunological environment in a way that confers a survival advantage for the subsequent developmental stages. The parasites could be exploiting the inherent mechanisms in place in the lung to regulate inflammation (25, 35). The regulatory mechanisms initiated in the lung might modulate the magnitude and the quality of subsequent immune responses against the parasite. In support of this idea are reports that a helminth infection modifies the immune responses to a number of pathogen and nonpathogen challenges (34, 43, 45, 76). Indeed, pathogen-induced counterregulatory mechanisms in the lungs are a basic tenet of the hygiene hypothesis (75, 78).
To our knowledge this is the first report on the histopathology of the lung phase of N. brasiliensis infection in mice. In WT BALB/c mice, there was a remarkably rapid resolution of mechanical damage and inflammation caused by N. brasiliensis larvae migrating through the pulmonary environment (Fig. 1). Within hours of the larvae exiting the lungs, the hemorrhage and inflammatory infiltrate were largely resolved, and by day 8 p.i., the histological appearance of the parenchyma of the lung and the large airways was nearly indistinguishable from that for uninfected controls (see inserts in Fig. 1). This rapid resolution took place in the context of cytokines IL-4 and IL-13, which have been demonstrated to mediate inflammation, epithelial cell hyperplasia, and mucus production in the lung (reviewed in reference 74). In contrast, the larva-induced inflammation in the lungs of SCID animals persisted through day 8 p.i., with significant levels of cellular infiltrate evident throughout the lung (Fig. 1H). The mechanistic basis for this difference in the ability to resolve pulmonary inflammation between WT and SCID animals is not clear but presumably reflects a defect in lymphocyte-mediated homeostatic mechanisms in the SCID animals.
Comparing the expression profiles from the lungs of infected WT and SCID mice provided an opportunity to study the complexities of the immediate innate immune response to N. brasiliensis larvae, to characterize the transition between the innate and adaptive immune mechanisms, and to infer the role of T cells in sustaining responses initiated by the innate immune system. Although there were differences, the dynamics and magnitude of the transcriptional and cellular responses in the lungs from WT and SCID mice associated with innate immune responses were similar during the first 4 days of infection. In the SCID mice, there was a rapid decay in many of the N. brasiliensis-induced cellular and transcriptional responses after day 4 p.i. (Table 1; Fig. 5C); however, transcription of a few genes, such as those encoding IL-10 and IL-13 (Fig. 4), as well as the number of alveolar macrophages (Fig. 5B), remained elevated. In contrast, a significant percentage of the transcriptional and cellular responses initiated during the innate phase were maintained or enhanced in the lungs of WT mice. The results underscore a complex regulation of inflammation in the lungs, with certain aspects under control of T cells while other facets are regulated in a T cell-independent fashion.
One of the most marked transcriptional changes observed in both WT and SCID animals was the immediate upregulation of the genes associated with AAM: ym1, ym2, fizz1, and arg1. Resident macrophages in different tissues adapt to their local environments based on interactions with secreted factors, surface signals from neighboring cells, and the nature of the extracellular matrix. Once established in the tissues, macrophages can be activated in a variety of ways. Classical macrophage activation occurs through stimulation from lymphocyte-derived IFN- and is key for the development of effective cell-mediated immune responses against intracellular pathogens (12). An alternative pathway of macrophage activation has also been defined that is mediated by IL-4 and/or IL-13 (22). IL-4/IL-13 activation of macrophages upregulates the mannose receptor (16), class II major histocompatibility complex, and CD80/CD86 (13) on their surfaces. It is generally thought that AAMs function in debris scavenging, tissue remodeling, wound healing, and the promotion of Th2 immune responses (22, 49, 63). It has also been demonstrated that AAMs inhibit T-cell proliferation (37, 41). The mechanisms through which AAMs carry out these functions are not clear. However, AAMs secrete a characteristic set of molecules, including YM1 (38), FIZZ1, and arginase (51), that are likely to contribute to the function of these cells.
YM1 is a member of a family of proteins that share sequence similarity to chitinases from lower organisms (29). Transcripts encoding YM1 can represent over 10% of the total nematode-induced AAM mRNA (38). YM1 has the ability to bind to chitin and related glycan structures (8), but its apparent lack of demonstrable chitinase activity makes its role as an effector molecule unclear. YM1 might play a defensive role by binding chitin-containing pathogens, such as fungi. for subsequent recognition by immune cells. Through its ability to bind to the extracellular matrix, YM1 might also mediate cellular trafficking that promotes wound healing and matrix repair—one of the proposed functions of AAMs (24, 59).
The functional significance of FIZZ1 is also yet to be defined. FIZZ1 (found in inflammatory zone I; also called resistin-like molecule ) was initially described as a product found in the bronchoalveolar lavage fluid of mice challenged with an allergen that was capable of inhibiting nerve growth factor in vitro (26). Subsequently it has been demonstrated that FIZZ1 is produced by macrophages in response to stimulation by IL-4 (38, 53, 57). FIZZ1, like YM1, has been implicated in regulating the deposition of extracellular matrix in the lungs (36), thus linking these two molecules to a role in pulmonary repair and remodeling. Also consistent with the previous report, we observed only a minor increase in the transcription of fizz2 in the lungs after N. brasiliensis infection (see Table S2 in the supplemental material). fizz2 appears to be preferentially expressed in the intestine (53, 64).
The balance between inducible nitric oxide synthase (iNOS) and arginase 1 is a central feature in the development of functionally distinct macrophage populations. iNOS and arginase 1 compete for L-arginine to catalyze the production of NO/L-citrulline or urea/L-ornithine, respectively. IL-4/IL-13 stimulate the production of arginase 1 and inhibit iNOS, while IFN- inhibits arginase and promotes iNOS production (24, 50). In the Th2 environment induced by N. brasiliensis infection, there was no increase in the expression iNOS (data not shown). Like YM1 and FIZZ1, arginase 1 is associated with repair and remodeling of tissues, specifically with collagen deposition and fibrogenesis (51).
Previous studies have shown that the induction of AAM-associated genes is dependent on IL-4 or IL-13 (22, 41, 73). The timing of the response in WT mice and the presence of the response in SCID mice indicate that T cells were not the source of these Th2 cytokines. The most likely source for this rapid production of IL-4 and IL-13 in the lungs is granulocytic cells—eosinophils, mast cells, or basophils (20, 62). Eosinophils, mast cells, and basophils have the capability of uncoupling transcription and translation of IL-4 and IL-13. These cells have been shown to constitutively transcribe IL-4 and IL-13 to form a reservoir of transcripts that can be mobilized for immediate cytokine production upon stimulation (20). Once activated, these cells can then continue to transcribe and produce IL-4 and IL-13. This ability to maintain cytokine synthesis after activation might explain the sustained production of IL-13 in the lungs of SCID animals (Fig. 4B). The significant increase in the transcription of IL-4 and IL-13 in WT lungs after day 4 p.i. is presumably due to T-cell-mediated adaptive responses.
The dynamics of IL-4 and IL-13 transcription differed significantly in WT and SCID lungs after N. brasiliensis infection (Fig. 4B). In the lungs from SCID animals, there was an immediate and selective upregulation of IL-13 transcription after N. brasiliensis infection that was not seen in WT animals. The cellular and molecular basis for this differential regulation between IL-13 and IL-4 is not clear (Fig. 4B), but it is likely to reflect fundamental differences between WT and SCID animals in the numbers and activation status of the cells in the lung that respond to innate signals. The cellular infiltrate induced by N. brasiliensis at days 2 and 4 p.i., measured by bronchial alveolar lavage, was dominated by macrophages, with only minor increases in eosinophil and neutrophil numbers (data not shown). The lack of significant changes in the transcription levels of eosinophil- and mast cell-associated genes in the expression profiling of N. brasiliensis-infected animals (data not shown) also indicated there was no major influx of these cells into the lungs. Recently, it has been demonstrated that basophils are a major source of IL-4 and that the number of IL-4-producing basophils in the lungs is increased 50-fold by N. brasiliensis infection (47). This increase in basophil levels is T cell dependent. It is possible that the lack of IL-4 production in the lungs of SCID mice reflects the defect in T-cell-mediated recruitment of basophils to the lungs. The major immediate cellular source of IL-13 in SCID lungs is yet to be determined.
AAMs have been shown to be induced outside the lung by other nematode infections (29, 37, 41, 53), as well as by digenetic trematodes (15, 18, 23), cestodes (7, 58), and protozoa (27, 57). The IL-4-dependent AAMs induced in the peritoneal or pleural cavities by the filarial nematodes Brugia malayi and Litomosoides sigmodontis, respectively, exerted a profound, contact-dependent antiproliferative effect on a range of different cell types, including antigen-specific T cells (37, 41, 67). Consistent with these previous observations, the AAAMs from N. brasiliensis-infected animals expressed class II major histocompatibility complex and readily took up large amounts of antigen but suppressed antigen-specific T-cell proliferation (data not shown). The mechanism through which AAMs suppress cell proliferation has not been defined. AAAMs could reflect a homeostatic mechanism in the lungs that functions to suppress inflammation after certain antigenic challenges, especially those that induce strong Th2 responses.
One of the complicating factors in assessing the innate response to N. brasiliensis is the inability to discriminate between the responses to parasite somatic and secreted antigens and the response induced by the focal mechanical damage caused by larval migration through the lungs. While it is not possible to evaluate the relative contributions of these two facets of larval invasion with the experimental design presented here, a survey of reports on the immediate transcriptional responses associated with chemical- or ventilator-induced acute lung injury show significant differences in the gene expression profiles from that induced by N. brasiliensis infection (39, 44, 46). While there are expected overlaps in the expression of genes involved in protection against oxidant injury, cell proliferation, and extracellular matrix repair, there is a notable lack of expression of genes associated with a Th2 response and AAM activation in the lungs of mice undergoing acute lung injury. Further experiments are required to determine the relative roles of parasite antigens and mechanical disruption to the signature expression profile induced by N. brasiliensis infection.
Immunohistochemical analysis of lungs from uninfected animals showed that YM1 was constitutively produced at modest but detectible levels by a subset of the GSL I+ alveolar macrophages (also F4/80+), which is consistent with a previous report of YM1 production in the lungs of adult mice (29). This constitutive production of YM1 in the lungs was supported by the intensity values of the transcriptional data (see Table S4 in the supplemental material). In addition, genes encoding other AAM-associated molecules, such as FIZZ1, MRC1, and AMCase but not Arg1, were also expressed in noninflamed lungs (see Table S4 in the supplemental material). These observations suggest that 15% of the cells typically referred to as alveolar macrophages are AAAMs (Fig. 5C). It is possible that the proportion might even be higher. One interpretation of the rapid conversion of the lungs from an environment that is populated with macrophages which are predominantly GSL I+/YM1– to an environment that is dominated by GSL I+/YM1+ cells is that the alveolar macrophages were directly converted to the AAAM phenotype as part of the innate response to N. brasiliensis. It is interesting to speculate that alveolar macrophages are incipient AAAMs that require innate or adaptive signals to fully mature. Like AAM, alveolar macrophages are commonly characterized as anti-inflammatory and have been shown to participate in tissue repair and remodeling (55). It is also possible that there is a rapid efflux and influx of cells into the lungs and that a majority of the cells that enter the lungs are AAM. Additional work is needed to define the trafficking of macrophages into and out of the lungs during inflammation and how the dynamics of trafficking influences macrophage populations.
.
ACKNOWLEDGMENTS
This work was supported by grants from NIH NHLBI (U01 HL66623) and NIH training grant T32AI007417.
We thank Joseph Urban (USDA, Beltsville, MD) for providing Nippostrongylus brasiliensis, Anne Jedlicka (Johns Hopkins Malaria Research Institute Gene Array Core Facility) for assistance in the gene array experiments, and Brian Schofield (Johns Hopkins University) for his assistance with immunohistocytochemistry.
FOOTNOTES
Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205. Phone: (410) 955-3430. Fax: (410) 955-0105. E-mail: ascott@jhsph.edu.
Supplemental material for this article may be found at http://iai.asm.org/.
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