Toll-like receptor 4 (TLR4) is required for protective immunity to larval Strongyloides stercoralis in mice

Microbes and Infection 9 (2007) 28e34 www.elsevier.com/locate/micinf

Original article

Toll-like receptor 4 (TLR4) is required for protective immunity to larval Strongyloides stercoralis in mice Laura A. Kerepesi a, Jessica A. Hess a, Ofra Leon a, Thomas J. Nolan b, Gerhard A. Schad b, David Abraham a,* b

a Department of Microbiology and Immunology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA Department of Pathobiology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104, USA

Received 27 July 2006; accepted 4 October 2006 Available online 6 December 2006

Abstract TLR4 is important for immunity to various unicellular organisms and has been implicated in the immune responses to helminth parasites. The immune response against helminths is generally Th2-mediated and studies have shown that TLR4 is required for the development of a Th2 response against allergens and helminth antigens in mice. C3H/HeJ mice, which have a point mutation in the Tlr4 gene, were used in this study to determine the role of TLR4 in protective immunity to the nematode Strongyloides stercoralis. It was demonstrated that TLR4 was not required for killing larval S. stercoralis during the innate immune response, but was required for killing the parasites during the adaptive immune response. No differences were seen in the IL-5 and IFN-g responses, antibody responses or cell recruitment between wild type and C3H/HeJ mice after immunization. Protective immunity was restored in immunized C3H/HeJ mice by the addition of wild type peritoneal exudate cells in the environment of the larvae. It was therefore concluded that the inability of TLR4-mutant mice to kill larval S. stercoralis during the adaptive immune response is due to a defect in the effector cells recruited to the microenvironment of the larvae. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: TLR4; Strongyloides stercoralis; Neutrophil; Nematode; T cell; Antibody

1. Introduction Strongyloides stercoralis is an intestinal nematode of humans causing disease in normal patients and potentially fatal disseminated strongyloidiasis in immunocompromised individuals [1]. Little is known about the protective immune response against S. stercoralis in humans, but infection is generally characterized by the development of a Th2 immune response with increased parasite-specific IgE and IL-4 production [2]. Coinfection with S. stercoralis and HTLV-1, a Th1-inducing pathogen, has been associated with downregulation of S. stercoralis-specific IgE and IL-5 responses [3], possibly as a result of increased IFN-g production due

* Corresponding author. Tel.: þ1 215 503 8917; fax: þ1 215 923 9248. E-mail address: [email protected] (D. Abraham). 1286-4579/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2006.10.003

to HTLV-1 infection [2]. The shift from a Th2-mediated response towards a Th1-biased response may be responsible for the high incidence of S. stercoralis hyperinfection in individuals infected with HTLV-1. In mice, protective immunity against the third-stage larvae (L3) of S. stercoralis requires the induction of a Th2-mediated immune response. In the absence of the Th2-associated cytokines IL-4 and IL-5, protective immunity does not develop [4,5], while intentional skewing of the immune response towards a Th1 response with rIL-12 results in loss of protective immunity [5]. Induction of protective immunity requires the presence of eosinophils during immunization but not during the challenge infection [4]. Additionally, larval killing during the adaptive immune response requires complement component C3 [6], IgM [7,8], and neutrophils [8,9]. Toll-like receptors (TLRs) are involved in recognition of invading pathogens via pathogen-associated molecular patterns

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(PAMPs) during both the innate and the adaptive immune responses [10]. There are currently 12 known TLRs [11], including RP105 [12]. One such receptor, TLR4, is required for signaling by lipopolysaccharide (LPS). Stimulation of antigen-presenting cells with LPS leads to the production of IL-12 [11], thus, TLR4 has been typically associated with the induction of a Th1 immune response. TLR4 has also been shown to be important for the induction of Th2-mediated allergic asthma responses in mice [13] and for the ability of dendritic cells to acquire a DC2 phenotype after pulsing with a helminth antigen [14]. TLR4 is expressed on B cells and regulates B cell responses to LPS [12]. In addition, mice deficient in TLR4 are impaired in their ability to recruit neutrophils [15,16]. Finally, mice deficient in TLR4 have been shown to have either decreased [17,18], increased [19] or unaffected [20] immunity to nematode infections. The objective of this study was to determine if TLR4 was required for protective immunity to larval S. stercoralis in mice. C3H/HeJ (TLR4-mutant) mice, which have a point mutation in the Tlr4 gene rendering them hyporesponsive to LPS [21], were used in these experiments. The requirement for TLR4 in the function of T cells, B cells and neutrophils was assessed. 2. Materials and methods 2.1. Animals and parasites C3H/HeJ (TLR4-mutant) and wild type C3H/HeOuJ were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in filter-top microisolator boxes under light- and temperature-controlled conditions. Male mice, 6e 8 weeks of age, were used for all experiments. S. stercoralis L3 were obtained from the fresh stools of a laboratory dog infected with the parasite according to methods previously described [22]. Larvae were collected from 7 day charcoal fecal cultures, washed and resuspended in a 1:1 mixture of NCTC-135 (Sigma) and IMDM (Sigma) with a mixture of 100 U of penicillin and 100 mg of streptomycin (Gibco, Grand Island, NY) per ml and 25 mg of levofloxacin per ml (Ortho-McNeil Pharmaceutical, Raritan, NJ). The medium from the last wash was incubated at 37  C overnight and no bacterial growth was observed. However, the medium from the last wash tested positive for the presence of LPS using a Limulus amebocyte lysate (LAL) assay test kit (BioWhittaker, Walkersville, MD). Construction of diffusion chambers and challenge infection of mice with L3 followed previously described methods [22]. Briefly, 14 mm Lucite rings (Millipore, Bedford, MA) were covered with 2.0 mm or 0.1 mm pore-sized Isopore membranes (Millipore). The membranes were attached to the rings with cyanoacrylate adhesive (Super Glue, Hollis, NY) and the Lucite rings cemented to each other with a mixture consisting of equal parts of 1,2 dichloroethane (Fisher Scientific, Pittsburgh, PA) and acryloid resin (Rohm and Hass, Philadelphia, PA); the completed diffusion chambers were sterilized in 100% ethylene oxide. L3 were injected into a diffusion

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chamber which was subcutaneously implanted on the back of the mouse. 2.2. Immunization and challenge with S. stercoralis L3 Mice were immunized at the nape of the neck on days 0 and 14 with 100 S. stercoralis L3. On day 21, mice were challenged with 50 S. stercoralis L3 contained within diffusion chambers. Implanted diffusion chambers were recovered on day 22 and live larvae were counted. Viability of larvae was determined by motility and morphology. Cells found within diffusion chambers were centrifuged onto slides using a Cytospin 3 (Shandon, Pittsburgh, PA) and then stained for differential counts with DiffQuik (Baxter Healthcare, Miami, FL). 2.3. Soluble L3 antigen preparation To clean the surface of the parasites and to remove debris from the larval cultures, L3 were incubated in a 1% solution of low melt agarose (Type I-A, Low EEO; Sigma). After the agar had solidified, PBS supplemented with antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.1 mg/ ml gentamicin) was added and larvae that migrated into the PBS were harvested and stored at 80  C. The L3 were defrosted, homogenized on ice in the PBS solution supplemented with a protease inhibitor cocktail (Sigma) with a final concentration of 10 mM EDTA, and then sonicated for 10 min. Deoxycholate (DOC; Sigma) soluble L3 antigens were obtained through a two-step purification procedure. PBS-soluble proteins were extracted first, followed by extraction of detergent soluble proteins from the remaining PBSinsoluble pellet done with 20 mM TriseHCl/0.5% DOC in the presence of protease inhibitor cocktail (Sigma). The solubilized proteins were dialyzed overnight against PBS, followed by sterilization through a 0.2-mm syringe filter. Protein concentration was determined by Micro BCA protein assay (Pierce, Rockford, IL). 2.4. Spleen cell stimulation Spleens from na€ıve and immunized mice were aseptically removed 1 week after challenge and made into single cell suspensions. Cells were cultured in a 96-well plate at 2  106/ well. Spleen cells were stimulated with PBS-soluble larval antigens added to a final concentration of 50 mg/ml. Plates were coated with anti-CD3 mAb at 0.5 mg/ml for 2 h at 37  C and washed with PBS. Cells were incubated at 37  C for 3 days and supernatants were collected and frozen at 20  C. 2.5. Parasite-specific IgM and cytokine ELISAs Nunc Maxisorp 96-well plates (Nunc, Naperville, IL) were coated overnight at 4  C with 50 ml of DOC-soluble L3 antigens at 10 mg/ml. Plates were blocked with borate blocking buffer solution (BBS; 0.17 M boric acid, 0.12 M NaCl, 0.05% Tween 20, 0.25% BSA, 1 mM EDTA, pH 8.5) at 37  C for 1 h. Wells were washed with distilled water and

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2.6. Passive transfer of immunity Serum from na€ıve and immunized mice was pooled and filter sterilized using a 0.22-mm syringe filter. Each serum recipient mouse was injected at the time of challenge with 100 ml of serum, brought up to 200 ml with 100 ml of PBS, in the subcutaneous pocket surrounding the implanted diffusion chamber. Parasite survival in the diffusion chamber was assessed after 24 h. 2.7. Peritoneal cell transfer to diffusion chambers Peritoneal cell recruitment was induced by intraperitoneal injection of 1 ml of 10% thioglycollate medium, brewer modified (Becton Dickinson, Sparks, MD) into wild type and TLR4-mutant mice. After 12 h, a peritoneal lavage was performed and the recovered cells were washed with sterile PBS and resuspended in the same medium as the larvae. Cells (3.4 million), either wild type or TLR4-mutant, were placed into diffusion chambers with L3. Diffusion chambers used for these experiments were constructed with 0.1-mm pore-size membranes to prevent resident cells from entering and experimental cells from exiting the diffusion chamber. 2.8. Statistical analysis

3. Results 3.1. Innate immune response against S. stercoralis in TLR4-mutant mice To evaluate the requirement of TLR4 in the innate immune response to S. stercoralis, diffusion chambers containing larvae were implanted in na€ıve mice for 3, 5 and 7 days. Larval survival was equivalent in the wild type and the TLR4-mutant mice at all three time points (Fig. 1A). In addition, the composition of cells recruited into the diffusion chambers did not differ between wild type and TLR4-mutant mice at any of the time points (Fig. 1B). These results indicate that TLR4 is not required for initiating larval killing mechanisms during the innate immune response. 3.2. Adaptive immune response against S. stercoralis in TLR4-mutant mice TLR4-mutant mice were immunized with L3 and challenged with L3 contained within diffusion chambers. Immunized wild type mice developed a protective immune

A

100

Larval Survival (%)

serum samples diluted in BBS were placed in duplicate wells at serial dilutions and incubated at 37  C for 2 h. Appropriately matched biotinylated goat anti-mouse IgM (Vector Labs, Burlingame, CA), IgG1 and IgG2a (Pharmingen) Abs were added and plates were incubated at 37  C for 2 h. Plates were washed and Extravidin peroxidase (Sigma) added for 30 min at RT, followed by the peroxidase substrate 2,20 azinodi(3-ethylbenzthiazoline-6-sulfonate) (ABTS) (Kirkegaard & Perry Laboratories, Gaithersburg, MD). ABTS color reaction was measured at 410 nm on a Dynatech MR5000 microplate reader. Cytokine ELISA for IL-5 was done using appropriately matched anti-IL5 monoclonal antibodies (Pharmingen) for coating and capturing antibody and the protocol was followed as above. An IFN-g kit (AN-18 monoclonal; Pharmingen) was used following the manufacturer’s provided protocol. 3,30 ,5,50 -Tetramethylbenzidine (TMB) was used as the substrate, the reaction was stopped using 0.5 M H2SO4 and color reaction measured at 450 nm.

80 60 40 20 0 3 - Day

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Experiments consisted of five mice per group unless otherwise noted. All experiments were performed at least twice, results were reproducible and data shown are from one representative experiment. Statistical analysis of the data was performed using MGLH multifactorial ANOVA with Systat version 11 software (Systat, Evanston, IL). Fisher’s least-significant-difference test was performed for post hoc analyses. Probability values of less than 0.05 were considered statistically significant.

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Fig. 1. Larval survival in na€ıve wild type and TLR4-mutant mice. Larval survival was equivalent in wild type and TLR4-mutant mice after being implanted subcutaneously in diffusion chambers for 3, 5 and 7 days (A). The number of neutrophils (N), macrophages (M), eosinophils (E) and lymphocytes (L) infiltrating into the diffusion chambers did not differ between wild type and TLR4mutant mice at all three time points. Data represent the means and standard deviations from five mice per group.

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Fig. 3. Analysis of the cytokine responses after immunization of wild type and TLR4-mutant mice. Spleen cells (2  106/well) were stimulated for 3 days with medium or soluble L3 antigens and cytokines measured from culture supernatants. (A) IL-5; *p  0.05 statistical difference between IL-5 levels in control mice infected for 24 h and immunized mice. (B) IFN-g; *p < 0.05 statistical difference between IFN-g levels in wild type and TLR4-mutant mice. Data shown represent the means and standard deviations of three pools of mouse spleen cells.

60

0

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IFN- γ ng/ml

response resulting in a 66% reduction in larval survival whereas immunized TLR4-mutant mice had no reduction in larval survival compared to na€ıve controls (Fig. 2A). The composition of cells recruited into the diffusion chambers implanted in immunized wild type and TLR4-mutant mice did not differ (Fig. 2B). Th1 and Th2 responses by spleen cells recovered from immunized wild type and TLR4-mutant mice were assessed. Th2 responses, as measured by IL-5 production after stimulation with soluble antigens from S. stercoralis, were elevated equivalently in immunized wild type and TLR4-mutant mice (Fig. 3A). Th1 responses, as measured by IFN-g production, were elevated in na€ıve and immunized TLR4-mutant mice as compared to their wild type controls. There were no significant differences, however, between the IFN-g responses of na€ıve and immunized TLR4-mutant mice. Treatment of spleen cells with the soluble antigens from S. stercoralis significantly reduced the IFN-g responses in all groups of mice relative to the background responses (Fig. 3B). Antibody responses were measured in the serum of immunized wild type and TLR4-mutant mice. Equivalent increases in parasite-specific IgM levels were seen in immunized wild type and TLR4-mutant mice (Fig. 4A). Parasite-specific

31

Macrophages

Eosinophils

600

IgG1 and IgG2a responses were not observed in the immunized mice from either strain. To confirm that the IgM response in the immunized wild type and TLR4-mutant mice was protective, passive transfer experiments were performed. Transfer of serum from immunized wild type or TLR4-mutant mice into na€ıve wild type mice resulted in equivalent reductions in parasite survival (Fig. 4B). 3.3. Wild type cells restore protective immunity to immunized TLR4-mutant mice

400

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Fig. 2. Larval survival in immunized wild type and TLR4-mutant mice. Protective adaptive immunity, as measured by larval survival, did not develop in immunized TLR4-mutant mice, as compared to immunized wild type mice (A). The type and number of cells infiltrating into the diffusion chambers did not differ between immunized wild type and TLR4-mutant mice. The number of neutrophils, macrophages and eosinophils infiltrating into the diffusion chambers did not differ between wild type and TLR4-mutant mice (B). Data represent the means and standard deviations of larval survival of five mice per group. Asterisks denote statistical difference ( p  0.05) between survival in na€ıve and immunized wild type mice.

Since there was no deficit in the protective IgM response or in cellular recruitment after immunization of TLR4-mutant mice, yet there was an absence of protective immunity in these mice, it was hypothesized that the defect in protective immunity was in the ability of the recruited cells to kill the larvae. PEC, consisting of approximately 70% neutrophils, 17% macrophages, 12% lymphocytes and 1% eosinophils, were elicited in wild type and TLR4-mutant mice. The cells were placed into 0.1-mm pore-size membrane covered diffusion chambers with the larvae. The 0.1-mm pore-size membranes prevent the influx and efflux of cells from the diffusion chamber, therefore allowing the direct analysis of the ability of a specific cell population to mediate larval killing in vivo. TLR4-mutant

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A

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Fig. 5. Peritoneal exudate cells from wild type mice restore protective immunity in immunized TLR4-mutant mice. Na€ıve and immunized TLR4-mutant mice were challenged with diffusion chambers containing only larvae (L3) or L3 with PEC that derived from either wild type (WT) or TLR4-mutant mice. The diffusion chambers were constructed with 0.1-mm pore-size membranes which prevent influx/efflux of cells from the chamber. Data represent the means and standard deviations. *p  0.05 statistical difference between larval survival in diffusion chambers containing only L3 compared to diffusion chambers containing L3 and PEC. **p < 0.05 statistical difference between larval survival in diffusion chambers containing L3 and mutant PEC compared to larval survival in diffusion chambers containing L3 and wild type PEC.

Immune

TLR4-Mutant

Fig. 4. Antibody response in immunized wild type and TLR4-mutant mice. Parasite-specific IgM titers (1:400 dilution) in serum did not differ between immunized wild type and TLR4-mutant mice (A). Passive transfer of serum from both immunized wild type and TLR4-mutant mice into na€ıve wild type mice transferred protective immunity (B). Data represent the means and standard deviations of larval survival of five mice per group. Asterisks denote statistical difference ( p  0.05) between values in na€ıve and immunized mice.

mice were immunized and then challenged with L3 alone or L3 with PEC from either wild type or TLR4-mutant mice implanted together in diffusion chambers. Immunized TLR4mutant mice challenged with diffusion chambers containing larvae and wild type PEC killed approximately 92% of the larvae, whereas 55% of the larvae were killed if the diffusion chambers contained TLR4-mutant PEC. Furthermore, na€ıve mice receiving wild type PEC had a statistically significant increase in their ability to kill larvae as compared to TLR4mutant PEC (Fig. 5). Therefore, these data suggest that the inability of immunized TLR4-mutant mice to eliminate challenge larvae was due to a defect in the ability of the cells recruited into the microenvironment to kill the larvae. 4. Discussion The objective of this study was to determine if TLR4 played a role in the development of protective immunity against larval S. stercoralis in mice. It was determined that C3H/HeJ mice, which have a mutation rendering signaling through TLR4 defective, do not develop adaptive protective immunity to larval S. stercoralis. This finding supports other reports suggesting that TLR4 may be important in the protective immune response to nematodes. Immunity against larval Brugia malayi, induced by injection of microfilariae, was

absent in C3H/HeJ mice as compared to the C3H/HeN wild type strain [17]. C3H/HeJ mice also did not develop protective immunity to the larval stages of Onchocerca volvulus following immunization with irradiated L3 [18]. Conversely, resistance to the adult gastrointestinal nematode Trichuris muris was enhanced in C3H/HeJ [19], suggesting that TLR4 may interfere with the development of a protective immune response to some nematodes. Finally, resistance to Syphacia obvelata is unaffected in mice deficient in TLR4 [20]. Therefore, based on these reports it is clear that the requirement in mice for TLR4 for the control of nematode infections varies based on the species and possibly the stage of the parasite. Protective immunity against S. stercoralis requires the induction of a Th2 immune response and is dependent on IL-5 [4,5]. It has been reported that TLR4-mutant mice do not develop a Th2 response to inhaled allergen in experimental asthma models [13] and the absence of the Th2 immune response was partially attributed to diminished dendritic cell function [13]. Therefore, it was hypothesized that the absence of adaptive protective immunity to S. stercoralis in TLR4-mutant mice may have been caused by a reduction in the parasite-specific Th2 immune response after immunization with S. stercoralis L3. Equivalent elevated IL-5 production by antigen stimulated spleen cells was observed for both wild type and TLR4-mutant mice after immunization. No differences were seen in the IFN-g production between control and immunized mice in either strain of mice. It was concluded that the antigen-presenting cells from the TLR4-mutant mice were capable of inducing a Th2 response against S. stercoralis, thereby suggesting that the failure of TLR4-mutant mice to develop protective immunity was not due to a defect in the T cell response. A similar observation was made in TLR4-mutant mice immunized with O. volvulus; protective immunity did not develop even though the mice

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generated elevated IgE, eosinophils and IL-5 responses [18]. This is in contrast to TLR4-mutant mice which developed increased resistance to T. muris coincident with increased Th2 responses [19]. The anatomical locations where S. stercoralis and O. volvulus L3 and T. muris adults reside are very different. Immunity to S. stercoralis and O. volvulus L3 is expressed in subcutaneous tissue, whereas immunity to T. muris adults, gut-dwelling nematodes, requires a mucosal immune response; therefore, the Th2-mediated immune mechanisms responsible for resistance are likely quite different. Both TLR4 and RP105 are expressed on B cells and regulate the B cell response to LPS [12]. In the absence of either receptor, the B cell response to LPS is severely impaired [12,23]. It was therefore hypothesized that the B cell response may be defective in the TLR4-mutant mice after immunization with S. stercoralis. Previous studies have demonstrated that protective immunity in mice to S. stercoralis is dependent on IgM [7,8]. The IgM response to S. stercoralis in immunized TLR4-mutant mice was similar to that of the wild type mice. Furthermore, passive transfer of serum from immunized TLR4-mutant mice to na€ıve wild type mice transferred protective immunity. Therefore, the induction of a protective antibody response against S. stercoralis L3 is TLR4-independent and differences in antibody production between wild type and TLR4-mutant mice do not explain the absence of protective immunity in TLR4-mutant mice. Neutrophils are required for killing larval S. stercoralis during the adaptive immune response [8,9]. Murine neutrophils express TLR4 [24] and TLR4 has been shown to be required for neutrophil mediated control of fungal [25] and bacterial [16] infections in mice. Mice deficient in TLR4 have been shown to have an impaired ability to recruit neutrophils [15,16], however, in the current study, there was no difference in cell recruitment for any cell type, including neutrophils, between wild type and TLR4-mutant mice. It was therefore hypothesized that the neutrophils recruited into the diffusion chambers in immunized TLR4-mutant mice were inherently defective in their ability to kill the larvae, based on previous reports of defective oxidative burst in neutrophils from TLR4-deficient mice [26]. To test this hypothesis, PEC from wild type and TLR4-mutant mice were implanted in diffusion chambers with larvae in immunized TLR4-mutant mice. PEC derived from wild type mice, transferred into immunized TLR4-mutant mice, killed the challenge larvae, thereby demonstrating that the defect in the protective immune response in the TLR4-mutant mice was in the function of effector cells. This conclusion was supported by the observation that transfer of PEC derived from TLR4-mutant mice into immunized TLR4-mutant mice did not reconstitute protective immunity to the same degree as the wild type mice. Eosinophils are required for the induction of the protective adaptive immune response to S. stercoralis in mice [4]. Since there was no defect in the induction of the adaptive immune response in TLR4-mutant mice, eosinophil function in TLR4-mutant mice appears to be intact. Both eosinophils and neutrophils can function as killer cells during the innate protective immune response in wild type mice [9]. Larval

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killing by the innate immune response in TLR4-mutant mice was equivalent to that seen in wild type mice. However, PEC recovered from TLR4-mutant mice had a defect in their ability to kill L3 in the innate immune response. This conclusion was based on the decreased larval killing efficiency of TLR4-mutant PEC transferred into na€ıve TLR4-mutant mice as compared to killing by PEC from wild type mice. The number of cells transferred into the diffusion chamber approximated the number seen in diffusion chambers recovered from immunized mice, which was approximately 10 times more than seen in diffusion chambers recovered from na€ıve mice. High numbers of PEC from wild type mice were capable of killing larvae in na€ıve mice, although not as effectively as in immune mice. PEC from TLR4-mutant mice, even at high numbers, were ineffective at killing larvae in na€ıve mice. There is therefore a discrepancy in that TLR4-mutant mice can effectively kill larvae in the innate response yet PEC from TLR4-mutant mice are defective in this capacity. A possible explanation is that in TLR4-mutant mice eosinophils compensate for the defect in the ability of neutrophils to kill in the innate response but cannot replace neutrophils in the antibody dependent adaptive response. In the cell transfer studies, significant numbers of eosinophils were not included in the diffusion chambers; therefore the defect in the neutrophils resulted in a reduction in larval killing. S. stercoralis L3 penetrate the skin to initiate infection after developing in fecally contaminated soil where gram-negative bacteria are present; therefore, it is probable that LPS is introduced to the host concurrently with infection with S. stercoralis. Furthermore, chronic infection with S. stercoralis is maintained by reinfection with autoinfective L3 that penetrate the mucosal walls [1], likely bringing with them gramnegative bacteria and/or LPS. The worms used in the current study contained residual levels of LPS even though the worms had undergone a thorough wash procedure. It is possible that the residual LPS may influence the activation state of the cells recruited into the microenvironment of the larvae during challenge infection. It cannot be concluded that LPS is the only molecule specifically signaling through TLR4 in this study, since molecules other than LPS, such as HSP60 [27], fibrinogen [28], and fibronectin [29], can activate cells via TLR4. Additionally, a protein related to the heat shock protein family was identified from Strongyloides venezuelensis [30] and a number of gene sequences from S. stercoralis are homologous with HSPs (NCBI nucleotide sequence database). Antigens from Schistosoma mansoni have been shown to signal through TLR4 [14], thus proteins from S. stercoralis larvae may have the ability to activate the immune response via TLR4 in an LPS independent manner. We therefore conclude that TLR4 is essential for adaptive protective immunity in mice to larval S. stercoralis. TLR4 is not required for T and B cell function but rather for activating neutrophils to mediate larval killing. Sufficient numbers of neutrophils are recruited in the absence of TLR4; however, the ability of the cells to collaborate with antibody to kill the worms was significantly reduced. The specific mechanism used by neutrophils to kill the larvae, that was absent from

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TLR4-deficient neutrophils, may be oxygen dependent, based on the reported deficiency in this pathway in TLR4-deficient neutrophils [26].

[15]

Acknowledgments

[16]

We would like to thank Ann Marie Galioto, Amy O’Connell, Udai Padigel and Kevin Redding for their expert and enthusiastic technical support. This work was supported in part by NIH grants RO1 AI47189 and RO1 AI 22662.

[17]

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