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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Cathelicidin-type antimicrobial peptides (CAMP) are important mediators of innate immunity against microbial pathogens acting through direct interaction with and disruption of microbial membranes and indirectly through modulation of host cell migration and activation. Using a mouse knock-out model in CAMP we studied the role of this host peptide in control of dissemination of cutaneous infection by the parasitic protozoan Leishmania. The presence of pronounced host inflammatory infiltration in lesions and lymph nodes of infected animals was CAMP-dependent. Lack of CAMP expression was associated with higher levels of IL-10 receptor expression in bone marrow, splenic and lymph node macrophages as well as higher anti-inflammatory IL-10 production by bone marrow macrophages and spleen cells but reduced production of the pro-inflammatory cytokines IL-12 and IFN-γ by lymph nodes. Unlike wild-type mice, local lesions were exacerbated and parasites were found largely disseminated in CAMP knockouts. Infection of CAMP knockouts with parasite mutants lacking the surface metalloprotease virulence determinant resulted in more robust disseminated infection than in control animals suggesting that CAMP activity is negatively regulated by parasite surface proteolytic activity. This correlated with the ability of the protease to degrade CAMP in vitro and co-localization of CAMP with parasites within macrophages. Our results highlight the interplay of antimicrobial peptides and Leishmania that influence the host immune response and the outcome of infection.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Endogenous antimicrobial peptides (AMPs) play an important role in innate immunity of organisms from all Kingdoms (Bardan et al., 2004; Bulet et al., 2004). AMPs attack and directly disrupt the surface membranes of microbes and in some cases work indirectly through modulation of host immunity (Lehrer et al., 1989; Powers and Hancock, 2003). A knock-out (KO) model of mammalian cathelicidin AMP (CAMP), the murine homologue of the human AMP LL-37, has been instrumental in establishing that this peptide is important for the control of microbial infections (Gallo et al., 1997; Nizet et al., 2001; Rosenberger et al., 2004; Lee et al., 2005). CAMP is expressed in epithelial cells, neutrophils and macrophages where it has antimicrobial activity but can be chemotactic to neutrophils, monocytes and lymphocytes (Brown and Hancock, 2006). CAMP expression in neutrophils is regulated by the proteolytic processing of a precursor protein to give rise to small C-terminal CAMP domain (Pestonjamasp et al., 2001). Aberrant host and microbial proteolytic modification of the CAMP domain can greatly affect its antimicrobial and immunomodulatory activity (Murakami et al., 2004; Sieprawska-Lupa et al., 2004; Braff et al., 2005a,b; Koziel et al., 2010).

Mammalian infection by the insect-borne parasitic protozoan Leishmania results in cutaneous, mucocutaneous or visceral leishmaniasis, which collectively afflicts 12 million people across 88 countries (Desjeux, 2001). Initiation of infection occurs by the delivery of flagellated parasites into the skin by infected insects. Parasites are engulfed by host phagocytes wherein they differentiate into aflagellated forms within phagolysosomes. Parasites withstand intracellular killing and replicate ultimately disrupting host cells. Various parasite surface glycolipids and proteins are important in Leishmania virulence such as a well-conserved group of cell-associated and secreted metalloproteases (termed leishmanolysins or gp63s) ofinsect and mammalian intracellular forms of Leishmania, which interface with innate immune components in early infection resulting in inactivation of complement and AMPs (Bouvier et al., 1995; Brittingham et al., 1995; McGwire and Chang, 1996; Joshi et al., 2002; Kulkarni et al., 2006). In mice, control of later stages of infection is dependent on a TH1 response and the production of IL-12 and INF-γ whereas progression of infection results from development of a TH2 response heralded by the production of IL-4 and IL-10 (Reiner et al., 1993; Reiner and Locksley, 1995; Rosas et al., 2005) There is partial overlap in the role of TH1 and TH2 cytokines in the immune responses controlling infection by different Leishmania species, but this is far from being completely understood (McMahon-Pratt and Alexander, 2004). IL-10 is produced by multiple cell types and works as an anti-inflammatory cytokine and is important to the control of cutaneous disease caused by New and Old World Leishmania (Kane and Mosser, 2001; Yang et al., 2007). Deficiency of IL-10 in Balb/c mice delays the accumulation of Leishmania mexicana and Leishmania amazonensis in lesions late in infection (Padigel et al., 2003). IL-10 may control NO production in infected macrophages and affect the elimination of intracellular parasites. Inversely reciprocal expression of IL-10 to that of IL-12 and INF-γ may be responsible for this effect (Kane and Mosser, 2001; Padigel et al., 2003).

While CAMPs are most likely critically important to the control of the host immune response in infection, studies on the modulation of pro- and anti-inflammatory cytokines have relied on exogenous administration of synthetic peptides to cells in culture (Bowdish et al., 2005; Mookherjee et al., 2006). Because the activity of exogenously administered and endogenous CAMP expression can have dramatically different effects (Pinheiro da Silva et al., 2009), it is difficult to extrapolate the in vitro data to endogenous activity. We have previously found that CAMPs have potent anti-leishmanial effects in vitro and leishmanolysins aids in partial protection of parasites by degrading and inactivating AMPs (Kulkarni et al., 2006; 2009). Despite our understanding of the mechanisms AMP-induced parasitic death in vitro (Kulkarni et al., 2009), the role of Leishmania–AMP interactions in vivo is not clear. Thus, we utilized the murine model to investigate the role of CAMP in control of cutaneous leishmaniasis. Further, we used the leishmanolysin knock-out line of Leishmania major to test the role of this protease in modulation of CAMP function (Joshi et al., 1998; 2002).

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Because CAMP is expressed by macrophages and is important in the control of bacterial skin infections (Nizet and Gallo, 2003; Bardan et al., 2004) we hypothesized that it may restrict the development of lesions produced by Leishmania. We compared the rate of lesion progression in wild-type (WT) and CAMPKO Balb/c mice infected with L. amazonensis, a New World cutaneous species (Fig. 1a). Lesions in the AMP knock-out line developed more rapidly than in the WT animals and the lesion size after 5 months were 3- to 5-fold larger in the animals lacking CAMP. We excised and explanted lesion tissue and quantified parasites by limiting dilution. Lesions harvested from the knockouts had 5-fold more parasites than those of WT animals (Fig. 1b). We also quantified parasites from local lymph nodes, various internal organs and bone marrow of all animals and found a slightly elevated, but not statistically significant, increase in parasites number from lymph nodes of CAMPKO animals. Conversely, there was significantly higher numbers of parasites in the spleen and bone marrow from the CAMPKO animals and little or undetectable parasites in those of WT controls. Analysis of explanted blood indicated the presence of parasites in 75–90% of CAMPKO animals whereas blood from 0% WT animals contained parasites.

image

Figure 1. CAMP is important for controlling lesion progression and parasite growth and dissemination of cutaneous L. amazonensis. a. Kinetics of lesion development, and; b. parasite burden in WT and CAMPKO (as indicated) Balb/c mice infected with 104L. amazonensis (LV78) for 5-months before lesion and organ harvest (n = 5). Data are means ± SD. One representative experiment of three is shown. P < 0.0001 for corresponding values in (a). P-values are shown above corresponding values in (b). Insets in panel (a) correspond to representative lesions of mice at 5-months of infection.

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We performed histopathologic examination of lesions and organs of infected mice to more closely determine the extent of parasite growth and the host cell inflammatory responses (Fig. 2). Lesions from WT mice demonstrated robust infiltration of inflammatory cells with the presence of large numbers of macrophages and neutrophils with limited numbers of visible parasites (Fig. 2a, upper panels) whereas, in stark contrast, lesions of mice lacking CAMP had minimal host inflammatory cells with large numbers of intra- and extracellular parasites (Fig. 2a, lower panels). Thus enhanced lesion development in mice lacking CAMP is accompanied by uncontrolled parasite growth in lesions and parasite dissemination into other organs. The presence of a robust inflammatory response in WT mice is inversely correlated with the size of, and parasite burden within, the lesions.

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Figure 2. Differential host cell infiltration and pro- and anti-inflammatory cytokine production between parasite-infected WT and CAMPKO mice. a. Histopathologic analysis of the tail-base lesions of WT and CAMPKO mice (lower magnification, 200×) infected with L. amazonensis reveals that the normal host cell infiltrate consisting of predominately neutrophils is absent in lesions of CAMPKO mice. Extensive numbers of parasites inside and outside of infected macrophages (black arrows in lower right panel) can be seen in comparison with WT mice in which few visible parasites are seen. b. Lymph nodes harvested from mice from animals in Fig. 1 were analysed histopathologically (left panels, 1000×) and for IL-12 and INF-γ production (right panels). Purple spots show infected macrophages containing parasites. c. Spleens harvested from mice were analysed histopathologically (left panels, 1000×) and for the IL-10 (upper right) and IL-12 (lower right). Purple spots show infected macrophages containing parasites (indicated by the black arrows) (n = 5 animals pooled). Data are means and ± SD from triplicate analyses. Similar data were obtained in three separated infection experiments. P-values are shown.

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Histopathologic analysis of lymph nodes and spleens of infected-CAMPKO mice revealed that they also had smaller infiltration of host immune cells (Fig. 2b and c, respectively). The differences in parasite burden between CAMPKO and WT animals were less dramatic in internal organs than in lesions reflecting the relatively less number of parasites reaching these sites. We analysed the levels of pro- and anti-inflammatory cytokines from pooled cells from organs in order to determine whether differences in these contribute to the differences in host cell response. ELISA assays using cytokine specific antibodies for IL-12, INF-γ, IL-4 and IL-10 were done using single cells suspension of pooled organs. In lymph nodes we found statistically significant (P ≤ 0.05) levels of pro-inflammatory cytokines IL-12 and INF-γ (Fig. 2b, right panel), each being approximately 4-fold higher in the WT than in CAMPKO animals. We found no differences in levels of anti-inflammatory cytokines IL-4 or IL-10 in these cells (not shown) between animal groups. Pooled splenic cells showed approximately 2-fold higher levels of IL-12 in WT than CAMPKO animals whereas the CAMPKO animals had ∼15-fold higher level of IL-10 than WT animals (Fig. 2c, right panels). There was no significant difference in IL-4 and IFN-γ levels within the spleens of animals (data not shown).

Because the anti-inflammatory cytokine IL-10 was found to be greatly elevated in CAMPKO mice we reasoned that this may be in part responsible for the lack of parasite control in these animals. Flow cytometric and RT-PCR analysis of IL-10 receptor expression revealed that macrophages in bone marrow, spleens and lymph nodes of CAMPKO mice had between 2- to 4-fold more IL-10R than WT animals (Fig. 3a and b). CAMPKO bone marrow macrophages infected in vitro with L. amazonensis produced significantly more IL-10 and had reduced levels of IL-12 than WT macrophages whereas they produced similar levels of TNF-α and IL-1β (Fig. 3c). This data strongly indicate that the lack of CAMP expression is important in the dysregulation of IL-10 secretion and IL-10R expression. Both of these factors probably contribute to the strong anti-inflammatory effect and exacerbation of parasite infection. IL-10 production by macrophages and/or T-cells and the presence of increased IL-10R probably provide a high level of anti-inflammatory activity leading to the deactivation of parasite infected macrophages. Whether CAMP is directly involved in tonal regulation of global IL-10 activity, by affecting IL-10 and/or IL-10R gene regulation or indirectly through regulating other cytokines or chemokines is unknown.

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Figure 3. Analysis of IL-10 receptor expression and cytokine production of WT and CAMPKO BMDMs. a. Fold increase of IL-10 receptor subunit expression from parasite-infected, IFN-γ or INF-γ + LPS stimulated BMDMs of CAMPKO over that of WT BMDMs. Analysis was done using qRT-PCR with β-actin as an internal control. The ratio of ΔΔCT values of CAMPKO over WT samples are shown with ± SD from triplicate experiments. Inset, flow cytometric analysis of surface expression of CD68 and IL-10R of BMDMs from WT and CAMPKO mice. b. Flow cytometric analysis of dual staining lymph node and spleens cells of WT and CAMPKO mice (as indicated). Flow cytometry experiments were done using macrophage-specific CD68-FITC (x-axis) and IL-10R-phycoerythrin (y-axis) antibodies. % of dual staining cells are indicated ± SD from triplicate experiments. c. Comparative ELISA of secreted cytokines (as indicated) by BMDMs of WT and CAMPKO mice infected with L. amazonensis in vitro. Infected BMDMs were left unstimulated or stimulated from 12–48 h with INFγ or INFγ + LPS (as indicated) and media were subjected to capture ELISA with cytokine specific antibodies. In the IL-12, IL-10 and IL-1β panels values for the 48 h time-point are given. Concentrations in pg ml−1 ± SD of three experiments are given.

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Proteolytic processing of CAMPs can alter their antimicrobial and immunostimulatory activities (Braff et al., 2005a). We have found that leishmanolysin can degrade a number of AMPs and greatly affect their activity (Kulkarni et al., 2006; 2009; McGwire and Kulkarni, 2010). Both insect stage and intracellular Leishmania express proteolytically active leishmanolysin that may potentially influence CAMP activity (Bahr et al., 1993; Ilg et al., 1993; Del Cacho et al., 1996; Streit et al., 1996; Hsiao et al., 2008) at early and late stages of infection, respectively. In order to investigate whether leishmanolysin affects CAMP function in vivo, WT L. major and a mutant line lacking leishmanolysin (Gp63KO) were compared in infection studies of WT and CAMPKO animals. This is the only Leishmania species with a complete knockout in leishmanolysin expression making it invaluable for the study of this protease in the context of parasite infection (Joshi et al., 1998; Joshi et al., 2002; Kulkarni et al., 2006; 2008; Lieke et al., 2008). Infection of foot-pads was allowed to progress over a 5-month period and then foot-pads and internal organs were harvested for analysis. Table 1 summarizes the differences in parasite burden found in various tissues. Explantation of foot-pad tissue and draining lymph nodes from WT mice infected with WT parasites had significantly more parasites than those infectedwith Gp63KO-parasites. Parasites from either line wereconfined to the foot-pads and lymph nodes of WT mice and could not be detected in distant organs. WT parasites were significantly more abundant in the foot-pads and lymph nodes of CAMPKO than in WT animals, consistent with the results described above for local replication of L. amazonensis. High numbers of mutant parasites were found in the lymph nodes, spleen and liver of all CAMPKO animals. Thus, the diminished virulence of the Gp63KO-parasites in WT animals imparted by the lack of leishmanolysin was obliviated in animals lacking CAMP expression.

Table 1.  Parasite burden of L. major infection of Balb/c mice.
 WTCAMPKO
FpLnSplLivFpLnSplLiv
  1. Five WT or CAMPKO mice (as indicated) per group were infected with indicated parasite lines via foot-pad and tissues were harvested at 5-months post-infection. Average parasite number [1+, 1–3/high-powered field (hfp); 2+, 4–6/hpf, 3+, 7–9/hpf, 4+, ≥ 10/hpf)] per mg of indicated tissue in explantation analysis.

  2. Fp, foot-pad; Ln, lymph nodes; Spl, spleen; Liv, liver.

  3. Parasites were not found in bone marrow or blood in these experiments.

Expt #1        
 LmjWT1+2+2+3+
 LmjKO1+4+4+3+2+
Expt #2        
 LmjWT1+2+2+3+
 LmjKO2+3+4+3+2+

Incubation of synthetic CAMP, in vitro, with WT- and Gp63KO-L. major lines indicates that the protease-deficient line is more susceptible to CAMP killing (Fig. 4a) and that the killing of both cell lines is dose-dependent. The enhanced susceptibility of the protease-deficient parasites is due to their inability to degrade CAMP (Fig. 4a, inset) as we have seen for other AMPs (Kulkarni et al., 2006). Because our infection data suggested that host CAMP and parasites interact in vivo and in vitro data showed that CAMP has the ability to associate directly with parasites, we analysed parasite-infected macrophages using anti-CAMP antibodies to determine if they interact directly (Fig. 4b and c). Immunofluorescence and confocal analyses clearly demonstrated the presence of extensive basket-like CAMP-staining structures surrounding parasites. Controls of uninfected cells and cells treated with secondary antibody alone did not show significant staining (not shown).

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Figure 4. Leishmania–CAMP association occurs within host macrophages. a. Dose-dependent activity of synthetic CAMP against WT- (white) Gp63KO-(black) L. major lines. Parasite survival was determined using an MTT metabolism assay and compared with untreated control reactions (Kiderlen and Kaye, 1990). Inset, SDS-PAGE analysis of full-length CAMP (10 µg) incubated in PBS for 24 h with glu-fixed L. major (107) lines (as indicated) showing degradation of CAMP by WT parasites only; degradation is inhibited with the gp63-specific inhibitor (not shown). b. WT L. major (top panels) or L. amazonensis (bottom panels) infected J774-line macrophages infected for 48 h subjected to IFA using α-CAMP antiserum (left panels) and co-stained with DAPI (middle panels) and merged (right panels) to visualize host- and parasite-DNA. c. Confocal microscopic visualization of Leishmania-infected J774-line macrophages for 24- (left side panel) and 72- (middle and right panels); Green fluorescence in left and right panels shows CAMP localization in parasitophorous vacuoles. Right panel, 5-,6-carboxyfluorescein diacetate succinimidyl ester-labelled Leishmania (green) infected macrophages were stained with α-CAMP antibodies and then detected with rhodamine-labelled anti-rabbit secondary antibodies (red). Orange areas demonstrate parasite-CAMP co-localization.

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To our knowledge this is the first report documenting the role of AMP in the control of parasitic infection in vivo. Our data support a model that CAMP is crucial for the local control of cutaneous lesion development and parasite growth and metastasis (Fig. 5). We surmise that CAMP expression increases within lesions attracting and activating host inflammatory cells and in doing so may act indirectly to limit parasite growth both by enhancing uptake of extracellular parasites by macrophages and neutrophils as well as acting on parasite-containing host cells to enhance parasiticidal activity. CAMP also may directly kill parasites in vivo by disruption of their cell membrane within the parasitophorous vacuole (McGwire et al., 2003; Kulkarni et al., 2006; 2009). In the absence of CAMP expression, local parasite growth within lesions remains uncontrolled and parasites can metastasize to visceral organs. CAMP expression correlates with the induction of pro-inflammatory and reduction in anti-inflammatory cytokines. IL-10 production and IL-10 receptor expression by the host, and particularly macrophages, appears particularly important to the exacerbation of infection. How CAMP is involved in maintaining a tonal level of IL-10 and IL-10R expression is unknown; however, there is data to support the link between expression of human LL-37 (the human CAMP homologue) and modulation of cytokine levels and function (Yu et al., 2007; Nijnik et al., 2009). CAMP expression and function is regulated by host proteolysis in vivo. Parasite metalloproteolytic action may serve to partially inactivate CAMP in WT animals giving rise to larger lesions with abundant parasites or may alter the function of the CAMP similar to aberrant host proteolysis which changes CAMP function leading to the enhancement of host inflammation (Koczulla et al., 2003; Yamasaki et al., 2007). This may be an additional way in which leishmanolysin contributes to parasite virulence. In the absence of CAMP expression this protease does not independently contribute, and even lessens, pathogenicity in this model. This work supports the idea that CAMP is a multifunctional host immune regulator with activity both early in infection, where it acts chiefly in innate immune responses, but also later in infection by directing adaptive immune responses by modulation of pro- and anti-inflammatory cytokines. Thus CAMP functionally bridges innate and adaptive immunity and microbial modulation of this host effector determines the outcome of infection.

image

Figure 5. Model of CAMP function during infection by Leishmania. Top panel, shows the intramacrophage Leishmania in parasitophorous vacuoles associating with CAMP (bicolour red and yellow), which can cleave CAMP into smaller fragments that may have alternate activities. CAMP may aid in intracellular killing of amastigotes (black). Extracellular CAMP peptides may act as a cytokine attracting uninfected host macrophages (MΦ), neutrophils and lymphocytes into inflammatory lesions. CAMP expression is associated with the upregulation (green arrows) of pro-inflammatory cytokines (shaded green) and downregulation (red arrows) of anti-inflammatory cytokines (shaded maroon) and IL-10R in spleen and lymph nodes (LNs). Expression of some cytokines is independent of CAMP expression (black arrows). Infected WT macrophages secrete IL-12, TNF-α, and IL-1β and low levels of IL-10 and express low levels of IL-10R on the surface membrane. Bottom panel, in the absence of CAMP expression there is a reversal of the expression of certain cytokines by spleen and LNs (note change in green and red arrows). Infected macrophages lacking CAMP expression are less activated and have a diminished ability to control intracellular parasite growth. Loss of repression of IL-10R expression occurs as does reversal in the secretion of certain cytokines; IL-10 is markedly increased and IL-12 is reduced. These MΦs have unaltered expression of IL-1β and TNF-α. In the absence of CAMP there is markedly less infiltration of host inflammatory cells into lesions, which contributes to more robust parasite growth and dissemination.

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Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Parasites

Parasites used in this study are: L. major (NHOM/SN/74/Seidman) and its Gp63KO-line (Joshi et al., 1998; 2002; Kulkarni et al., 2006) and WT L. amazonensis LV78 (McGwire and Chang, 1994; 1996). All strains were maintained routinely as insect forms in M199 (Invitrogen) containing 10% HIFBS.

Peptide and antisera

Synthetic mCAMP peptide (NH2-GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ-COOH) was purchased from AnaSpec (Fremont, California, USA) and used for in vitro assays. Antiserum against mCAMP was generated as previously described (Gallo et al., 1997; Nizet et al., 2001).

Animals

BALB/c mice were purchased from Harlan (Madison, WI, USA). The Cnlp−/− (CAMP knockout) (Nizet et al., 2001) mice were bred from CAMP knockout BALB/c breeding pairs at The Ohio State University. Mice have been backcrossed at least 70 times. All protocols were approved by the Institutional Animal Care and Use Committee at The Ohio State University.

Infection of mice

For infection with L. amazonensis; male mice (6–8 weeks old) were inoculated subcutaneously with 106 late stationary-phase promastigotes in 50 µl of Hepes-buffered saline at the dorsum of the tail-base. Lesion development was measured weekly with a Vernier caliper gauge. For L. major infection, mice infections were inoculated in the left hind foot-pad with 106 (in 10 µl Hepes-buffered saline) metacyclic WT or mutant L. major (Joshi et al., 1998). Foot-pad thickness was measured at weekly intervals with a Vernier caliper. Five mice were used in all treatment groups.

Quantification of parasites

Skin lesions, infected foot-pads, lymph nodes and organs were excised, weighed and rinsed with PBS then homogenized through 45 µm mesh barrier. Tissue was centrifuged at 1500 g for 15 min, washed with PBS then serially diluted in M199 + 10% HIFBS and observed daily for the transformation of amastigotes to promastigotes. Parasites were enumerated microscopically and expressed as no. per mg tissue.

Cytokine ELISA

Draining lymph nodes and spleens were excised under sterile conditions and single cell suspensions were prepared by gentle teasing through 45 µm mesh in complete RPMI 1640 (Invitrogen), supplemented with 10% HIFBS, 100 µ ml−1 penicillin-streptomycin and 2 mM glutamine. Red blood cells were lysed using Boyle's solution and viable cells counted using trypan blue exclusion and adjusted to 3–6 × 106 cells ml−1; 3 × 105 cells were plated into each well of 96-well flat-bottom plates and stimulated with 40 µg ml−1 of Leishmania soluble antigen for 72 h at 37°C. Supernatants were collected from these cultures, and the levels of IL-12, IFN-γ, IL-4, IL-10, TNF-α and IL-1β were measured by ELISA. All reagents for cytokine ELISA were purchased from BioLegend Inc. (San Diego, CA, USA). The cytokine concentrations were expressed in pg ml−1 ± SD of three separate experiments. All values were in the range of concentrations found under the same conditions with WT cells of the same subtype.

Histopathological examination

Mice were euthanized and excised lesions and tissues were fixed with 10% buffered-formalin and embedded in paraffin. Paraffin sections were stained with haematoxylin-eosin and examined under light microscopy. For each tissue 10–20 high-power fields were examined and representative fields were chosen for presentation.

Parasite survival assay

A standardized parasite survival assay was used as described previously (McGwire et al., 2003; Kulkarni et al., 2006). Briefly, 107 parasites were incubated in 100 µl of Medium 199 for 2 h at the indicated amount synthetic mCAMP peptide. MTT reagent was added to reaction and incubated overnight followed by treatment with 10% SDS for 6–8 h followed by reading in spectrophotometer at 570 nm. Treated parasites were compared with parasites incubated in the same conditions either in medium alone or in the presence of a non-AMP peptide at the same concentration.

Real-time PCR

Bone marrow-derived macrophages (BMDMs) from Balb/c WT and CAMPKO mouse were isolated and plated at 1 × 106 ml−1 in RPMI medium then infected with late stationary-phase L. amazonensis promastigotes at a parasite : BMDM ratio of 10:1 for 24 h after which non-engulfed parasites were removed by washing. Replicate infected BMDMs were left untreated or stimulated with INF-γ (100 U ml−1) or INF-γ with LPS (200 µg ml−1) for 12–72 h. Infected BMDMs were washed three times with PBS and total RNA was prepared using the Trizol RNA isolation kit (QIAGEN) according to the manufacturer's protocol. Synthesis of cDNA was completed with Reverse Transcriptase III using SuperScript VILO cDNA synthesis Kit (Invitrogen) according to the manufacturer's recommendations. Quantitative real-time PCR was performed on a Biorad CFX real-time PCR machine using SYBR green Universal PCR Master Mix (Biorad). All values were internally normalized to β-actin expression in the same cDNA set. Data are presented in the terms of fold increase in gene expression level and represented as the average of three experiments ± SD. The primer sequences used are as follows: IL-10Rα-subunit: forward primer, 5′-CCCATTCCTCGTCACGATCTC-3′ and reverse primer, 5′-TCAGACTGGTTTGGGATAGGTTT-3′. IL-10Rβ-subunit: forward primer, 5′-ACCTGCTTTCCCCAAAACGAA-3′ and reverse primer, 5′-TGAGAGAAGTCGCACTGAGTC-3′ and β-actin: forward primer, 5′-TCAGGCAGCTCATAGCTCTT-3′ and reverse primer, 5′-ACCGAGCGTGGCTACAGCTT-3′.

Flow cytometry

Single suspension BMDMs, lymph node cells and spleen cells were analysed by three-colour flow cytometry as described previously (Rosas et al., 2005). Briefly, 0.5–1 × 106 cells in 50 µl were incubated with prestaining buffer (PBS, 4% BSA, 0.5% sodium azide, 10% mixture of FCS and anti-Fc receptor antibody) for 10 min. Cells were then stained with phycoerythrin-conjugated anti-IL-10 and FITC-conjugated anti-CD68 (0.5 µg ml−1) for 30 min. The cells were washed twice, fixed in 1% formaldehyde and analysed with a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA, USA).

Immunofluorescence analysis

J774 mouse macrophages were grown on coverslips in 24-well plates (2 × 105 cells per well) and infected with late stationary-phase/metacyclic L. amazonensis or L. major parasites [in some experiments parasites were pre-labelled with 5-,6-carboxyfluorescein diacetate succinimidyl ester (Goncalves et al., 2005) for at 37°C for 24 h after which non-engulfed parasites were washed away and cultures were incubated further at 37°C. At different time points (24, 48, 72 h) coverslips containing infected macrophages were washed twice with PBS and fixed with methanol overnight. Fixed coverslips were rehydrated with PBS and blocked with 5% FBS, then incubated with a rabbit polyclonal α-CAMP antiserum (1:500), washed thrice with PBS and then probed with a 1:5000 dilution of either FITC- or rhodamine-conjugated anti-rabbit secondary antibody. Replicate slides incubated with either pre-immune rabbit serum or secondary antibody alone served as controls as did coverslips containing uninfected macrophages to determine baseline CAMP levels. Coverslips were mounted using ProLong Gold containing DAPI (Invitrogen) and sealed with nail polish. Slides were analysed using am Olympus Flowview 1000 Laser Scanning Confocal microscope.

Statistical analysis

Student's unpaired t-tests were used to determine statistical significance using a cut-off of P ≤ 0.05.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

This work was funded internally by faculty recruitment funds from The Ohio State University and indirectly with grants from the American Heart Association to B.S.M. We thank members of the Satoskar lab and Dr. Amal Amer for help with the cytokine analysis.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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