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Central European Journal of Immunology
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3/2003
vol. 28
 
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The immunopotentiating effect of Listeriolysin O in the response against Leishmania major in BALB/c mice

Jawaher A. Al-Salem
,
Khaled S. Tabbara

Centr Eur J Immunol 2003; 28 (3): 119–125
Online publish date: 2004/04/29
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Introduction


Listeria monocytogenes is a Gram-positive, facultatively intracellular bacterium, which invades and parasitizes macrophages, as well as a wide range of other cells, including mesenchymal and epithelial cells. Once internalized, L. monocytogenes escapes the phagosome, and multiplies in the cytosol of the parasitized cell. Escape from the phagosome is largely mediated by Listeriolysin O (LLO), a 58 Kd thiol-activated pore-forming cytolysin, which has been shown to be a major virulence factor in this organism. Immunization with LLO-producing, but not nonproducing strains induces protective T cell mediated immunity [1, 2]. The innate response to LLO-producing L. monocytogenes is associated with the production of several cytokines, including the type 1 cytokines IL-12 and IFN-γ [3–5], both of which are required for protective immunity [6, 7]. LLO itself appears to be a participant in this stimulatory activity, as shown by upregulation of expression mRNA of these cytokines following in vitro stimulation of spleen cells with purified LLO [8].

The protective immune response against L. major is dependent on the expansion of a population of IFN-γ-
-producing T lymphocytes, and the induction of Nitric Oxide (NO) – dependent killing within parasitized macrophages [9]. The cytokine milieu during the primary expansion of T lymphocytes is one of the major factors influencing the direction of Th cell polarization. Interleukin-12, a pro-inflammatory cytokine primarily produced by activated macrophages [10] and dendritic cells [11] has been shown to be central for the development of the protective Th1 response, while IL-4 is the major cytokine driving Th2 predominance and susceptibility [9].

The BALB/c mouse is highly susceptible to L. major, developing progressive non-healing lesions at the site of infection, followed by visceralization and death. The inability of this mouse strain to resist L. major is associated with its failure to develop the appropriate Th1 response. Events in the early stages of infection, including an early burst of IL-4 production resulting in the down-regulation of expression of the β2 subunit of the IL-12 receptor [12], and a breakdown of IL-12 production [13], appear to contribute to the predominance of a Th2 response. Manipulations resulting in increased or sustained IL-12 production, particularly during primary encounter with the parasite or its antigens, reverse this response leading to Th1 predominance and protection [14–16].

Given the predominance of type 1 cytokines (IL-12 and IFN-γ) produced during the early immune response to
L. monocytogenes, we have previously investigated the immunomodulatory effect of coinjection of an LLO-producing L. monocytogenes on the development of the immune response of BALB/c mice to L. major. In these studies (submitted for publication) [17], we showed the early polarization of the response against L. major towards a protective IFN-γ-dominated response resulting in a significant delay in lesion development, extended survival, control of parasite multiplication and dissemination. This response was associated with the upregulation of IFN-γ production and the downregulation of IL-4 protein and message expression in draining LN cells from coinjected mice. In this paper, and as a follow up to our earlier findings, we investigated whether the immunopotentiating role of L. monocytogenes in this model is related to LLO production.



Material and Methods

Mice


Female BALB/c mice raised and maintained at the animal facility of the Arabian Gulf University, from a stock originally obtained from Olac (U.K.), were used at 8–12 weeks of age. Mice were age-matched for each experiment.


Parasite preparation and footpad injections


A Leishmania major isolate (MOHM/SD/87ELD, isoenzyme LON1), originally obtained in 1987 from a cutaneous ulcer of a 50-year-old Sudanese male patient [18] was used throughout this study. Parasites were cultured at 22°C in Schneider’s medium (Sigma), supplemented with 10% heat inactivated fetal bovine serum (FBS) (ICN), 5mM HEPES, 50IU penicillin/ml (ICN), 50 µg streptomycin/ml (ICN), and 2% human urine (complete Schneider’s Medium). Cultures were refreshed every 2–3 months by re-isolating from infected footpads of BALAB/c mice to maintain virulence.

Stationary phase, four-day-old L. major promastigote cultures were used for injections. Promastigotes were collected by centrifugation, washed twice with Phosphate Buffered Saline (PBS), adjusted to 2x107/ml and 50 ml (1x106) was injected subcutaneously in the footpad using a 25-gauge needle. Footpad thickness was measured weekly using a Vernier Caliper. The contralateral footpad was measured as a control.


Listeria monocytogenes culture and footpad injections
Two Listeria monocytogenes strains were used: 1) an LLO-producing (LLO+) clinical isolate obtained from the pathology department at Salmaniya Medical Complex (Bahrain); and 2) an LLO-nonproducing (LLO-)
L. monocytogenes strain (NCTC 10357). L. monocytogenes stocks were maintained by monthly subculture on Brain Heart Infusion (BHI) agar plates.

Cultures for injection were prepared by inoculating 150 ml BHI broth with 1ml of an overnight culture. Growth at 37°C, on a rotary shaker, was monitored by hourly spectrophotometric readings at 600 nm. In preliminary experiments, the growth curves of both L. monocytogenes strains were determined and viable counts correlated to optical density (OD) at 600 nm. At the appropriate OD an aliquot of the culture was centrifuged and the pelleted organisms washed in cold PBS and adjusted to the desired concentration for injection. For coinjection experiments, organisms were mixed such that a 50 ml volume would simultaneously deliver the desired numbers of both organisms. Appropriate dilutions from the bacterial cultures used for injections were plated on BHI agar to ascertain actual viable counts.


Estimation of Parasite Load

Mice were sacrificed by cervical dislocation at various intervals following infection. The footpads, spleen and draining popliteal lymph nodes were aseptically removed. Footpads were de-boned and homogenized in cold HBBS using a glass homogenizer, while spleens and lymph nodes were mashed between the frosted ends of two glass slides to obtain single cell suspensions. Tissue suspensions were serially diluted in complete Schneider’s medium in flat bottom 96-well microtiter plates. Plates were placed in a humidified box and incubated at 26°C for 7 days. Positive wells (showing growth) were scored and the number of viable parasites, per organ, was calculated from the reciprocal of the highest positive dilution.


Stimulation of in vitro IFN-γ Production

Two million popliteal lymph node cells were stimulated in vitro with 1x106 live L. major promastigotes from a
4-day-old culture. Cells were cultured in a volume of 2 ml of RPMI 1640 (ICN) supplemented with 10% FBS, 25 mM HEPES, 50 IU penicillin/ml, 50 µg streptomycin/ml, 2 mM Glutamine, and 5 x 10-5 M 2-mercaptoethanol, in 24-well tissue culture plates. Supernatants were collected following 72hrs of culture, filtered and stored at -80°C until assayed.


Determination of IFN-γ Concentration

Interferon-γ concentrations in culture supernatants were determined by Enzyme Linked Immunosorbent Assay (ELISA) using a commercial kit (Pharmingen-U.S.A) following manufacturer’s procedure.


Purification of Listeriolysin O

Purification of Listeriolysin O was carried out following the procedure of Tsukada, et al. [19]. Briefly, 3 liters of BHI broth were inoculated with the LLO+ strain of L. Monocytogenes and incubated for 18–22 hrs at 37°C. The supernatant was collected by centrifugation and filtered using a 0.45 µm Millipore filter unit. Supernatant proteins were precipitated in the cold with 60% saturation of ammonium sulfate. The precipitate was solubilized in a volume of PBS equal to 15% of the original supernatant volume, dialyzed at 4°C overnight against two changes PBS and then against 25 mM Tris-buffer, pH 8.0. The dialysate was applied to a DEAE-Sephacel column (LKB) and eluted with a gradient of 0–0.5 M NaCl while maintaining the column at 4°C. 1ml fractions were collected on ice, assayed for hemolytic activity, hemolytic fractions pooled and concentrated 15–20 times in a Centricon centrifugal filter devices (10 kDa).


Sodium Dodecyl Sulfate – Polyacrylamide Gel electrophoresis (SDS-PAGE)

Culture supernatant proteins and semi purified hemolytic fraction concentrates were analyzed using vertical SDS-PAGE (Mini-protean II Electrophoresis Cell, Bio-Rad). Samples were boiled for 4 min in an equal volume of sample buffer consisting of 3.8 ml deionized water, 0.1 ml of 0.5 M Tris-HCl buffer (pH 6.8), 0.8 ml glycerol, 1.6 ml of 10% SDS, 0.4 ml of 2-mercaptoethanol and 0.4 ml of 1% (w/v) bromophenol blue. Treated samples were applied to a 12% polyacrylamide gel and electrophoresed at 200 V and 60 mA for 40 min. Protein bands were visualized by staining with Coomassie blue and silver stain. Molecular weight standards were run in parallel. Gels were dried at 50°C for 45 min, laminated and photographed.


Tissue Processing and Staining for Light Microscopy

Spleens specimen were fixed in 10% buffered formalin (pH 7.2), processed routinely for light microscopy and 5µm paraffin sections were stained with Hematoxylin and Eosin.


Results


Coinjection with an LLO-Producing but not an LLO-Nonproducing L. monocytogenes strain attenuates L. major lesion development in BALB/c mice



To evaluate the role of LLO in protection of BALB/c mice against L. major, mice were coinjected with 1x106 L. major plus an LLO+ or an LLO L. monocytogenes strain. A dose of 1x103 organisms, which was previously shown to be protective, was used for the pathogenic LLO+ strain. Since the LLO- strain lacks virulence, and are thus less replicative, a larger dose of 1x106 was used to simulate a similar antigenic dose, based on peak numbers attained by the virulent strain when 1x103 organisms are injected (results not shown).

The evolution of lesions, as measured by the increase in footpad thickness was monitored weekly. As shown in Figure 1, control group mice injected with 1x106 L. major, as well as groups coinjected with the LLO+ or LLO strains developed measurable lesions beginning week 4 following injection. Lesions size increased steadily over the following weeks in all three groups. The mean lesion size of mice coinjected with the LLO+ L. monocytogenes strain was smaller than those of the two other groups and the difference was statistically significant different (p<0.05) between weeks 4 and 8 following injection when comparing the group coinjected with the LLO+ strain to the two other groups. There was no difference in lesion size between mice coinjected with the LLO strain and those injected L. major only.

Differences among the three groups are further illustrated by the pattern of lesions developing. As shown in Figure 2, both the control group and the group coinjected with the LLO strain developed open, ulcerating lesions 8 weeks following injection. In contrast lesions of the group coinjected with the LLO+ strain were nonulcerative.


Coinfection with the LLO-producing L. monocytogenes strain reduces the parasite load


In order to determine the effect of LLO production on in vivo parasite multiplication, footpads and popliteal lymph nodes were removed at 3, 5 and 8 weeks and spleens at 5 and 8 weeks following infection with L. major only, or together with LLO+ or LLO L. monocytogenes. Two mice from each group were sacrificed at each time point. Footpads and spleens were assayed individually, while lymph nodes were pooled. The parasite burden of footpads from control mice and those coinjected with LLO L. monocytogenes followed a similar pattern. The averaged number of parasites per footpad of these two groups at the earliest point (3 weeks), when lesions were hardly measurable, was significantly increased (4x107) over the injection dose of 1x106 further increased to about 9x108 by week 5, then remained at a similar level by week 8. In comparison, the parasite load of footpads of mice coinjected with the LLO+ L. monocytogenes strain was 60 fold lower at week 3, and 400 fold lower at week 5, but approached similar levels to the two other groups at week 8 (Figure 3).

Parasites numbers were lower in the lymph nodes than at the site of injection. At 3 weeks, the parasite loads of the control group and the group coinjected with the LLO strain were 8x106 and 5x105 respectively. In comparison, the group coinjected with the LLO+ strain had a parasite load that was about 2.5 logs lower than the group injected with the LLO strain. However, by week 5, parasites increased in the LLO+ coinjected group to a level similar to that of the control group, while the LLO coinjected group was about one log higher. At the peak of lesion development, at 8 weeks, the parasite loads of both control and LLO coinjected groups were further increased while the group coinjected with the LLO+ strain remained essentially unchanged.

Differences in parasite loads of the three groups were most evident in the spleens. Based on earlier observations, which showed lack of visceralization before 4 weeks, the parasite load of spleens was only assayed at 5 and 8 weeks following injection. Parasites were detectable in spleens of the control and LLO- coinjected mice 5 weeks following challenge and increased by about one log at 8 weeks. In contrast parasites were not detected at both points in spleens of mice coinjected with the LLO+ strain (<102 per spleen).


Lymph node cells from mice coinjected with the LLO-producing L. monocytogenes strain produce higher levels of Interferon-γ

The IFN-γ response of LN cells to L. major promastigotes was monitored following injection of
L. major alone, or following coinjection with LLO+ and LLO L. monocytogenes strains (Figure 4). Two mice were sacrificed at each time point, lymph nodes pooled and stimulated in vitro. Low levels of IFN-g were detectable in all three groups as early at 3 weeks, peaked at 5 weeks, and then sharply decreased at the two following measurement points (weeks 8 and 10). The three groups of mice produced comparable levels of IFN-γ at 3 weeks. However, at 5 weeks, the group coinjected with LLO+ strain produced the highest level (13,350 pg/ml) followed by mice coinjected with the LLO- strain (10,700 pg/ml), while the control group produced significantly lower concentrations of IFN-g (2,525 pg/ml). While all groups produced lower levels of IFN-γ at 8 and 10 weeks, mice coinjected with the LLO+ strain were the dominant producers at both points (4,440 and 2,200 pg/ml Vs 1,075 and 320 for controls, and 1,330 and 1,275 for the group coinjected with LLO- strain).



Effects of Coinjection of semi-purified Listeriolysin O


Listeriolysin O was semi-purified from L. monocytogenes culture supernatants to a single band on SDS gel as revealed by Coomassie blue staining (Figure 5). Silver staining, which detects lower concentrations of protein, however, revealed multiple bands. Coinjection of 80 mg of semi-purified LLO into footpads of mice together with 1x106 L. major did not attenuate lesion development (data not shown). However, at peak lesion development, coinjected mice appeared less stressed than controls, which developed classical stress symptoms (ruffled fur, hunched back, sluggish movement). Nineteen weeks following injection, six mice were sacrificed from each of the two groups, and their spleens collected.

Spleens from LLO coinjected mice were significantly smaller than those of the control group (681±60 mg Vs 803±34 mg, p<0.001). Gross examination showed significant external granulomas on spleens from the control group, while only one of the six spleens from the coinjected group was grossly granulomatous. Histopathological changes in mice of the two groups (Figure 6) revealed significant differences. Spleens of mice injected with L. major alone developed extensive disorganized macrophage/monocyte granulomas with multinucleated giant cells, a very high parasite load, and occasional necrotic foci. In contrast, spleens from mice coinjected with LLO developed well-defined immature focal granulomas, in the red as well as the white pulp, with low parasite numbers and the absence of multinucleated giant cells and necrotic foci.


Discussion

The ability to produce LLO is unique to virulent L. monocytogenes, and only LLO-producing strains are capable of inducing the critical T cell-dependent response required for protective immunity against this pathogen. Although LLO is in itself an important target antigen for the stimulation of the CD4+ T cell response [20] its main role in the induction of the protective response appears to be as a bacterial modulin in the cytokine response rather than as a target antigen [21]. Through its action on macrophages, LLO stimulates the production of IL-12, which in turn drives NK cells to produce the macrophage-activating cytokine IFN- γ [8]. Together those two cytokines, produced by the innate response, drive the developing acquired T cell response towards an IFN- γ - dominated protective Th1 response.

Our results suggest that Listeriolysin O production in our system is also closely linked to protection against
L. major in that lesion evolution in mice coinjected with the LLO+ strain was significantly delayed and reduced and that the lesion displayed a less aggressive pathology, while coinjection with the LLO strain offered no such advantage. Coinfection with the LLO+ strain also clearly potentiated the L. major-specific IFN-γ response resulting in the control of parasites at the site of injection, the draining lymph nodes, and spleens. Of particular note was the significant delay of spread of the parasite to the spleen, in spite of significant parasite load at the site of injection and the lymph nodes at that time. It appears possible that a potentiated response limits visceralization though not able to entirely eliminate parasites at the injection site or to prevent spread to the local lymph node. The results suggest LLO produced by the virulent strain was essential for the generation of protective immunity, but the possibility that other cell products may be involved cannot be ruled out, particularly since the two L. monocytogenes strains utilized were not isogeneic.

A transient increase of in vitro IFN-γ response of mice coinjected with the LLO strain was noted at 5 weeks but returned later to levels comparable to those of mice challenged with L. major alone. It is possible that bacterial components, such as LPS, at the higher dose of injected LLO- strain, may have lead to this observation, although one would have expected this at a time point closer to injection.

The injection of semi-purified LLO was not as effective as the injection of LLO+ organisms. Although histological changes observed in the spleen including the containment of the parasite in focal areas with lower parasite loads, the preservation of normal histological features of the spleen and the absence of necrosis are consistent with protection, yet footpad lesion sizes were not affected. It is possible that various indicators of protection may not all be similarly affected. Soussi et al. [22] reported a similar observation where no reduction in lesion development was detected in BALB/c mice immunized with an LLO-producing attenuated (ActA mutant) L. monocytogenes expressing the LACK antigen (a major antigen) of L. major, although IFN-γ was specifically produced by spleen cells stimulated in vitro with L. major or the LACK antigen. Several possibilities may account for the observed lack of effect of LLO on lesion development. First, the amount of LLO injected may not have been sufficient to induce adequate macrophage stimulatory effects, particularly as our product was not pure, which would reduce the specific activity of the LLO preparation. In studies on the role of LLO in stimulating an anti L. monocytogenes response [23], immunization with double the quantity of crude LLO (of the one we used) achieved a reduction in bacterial load, which was 1000 times lower than immunization with live, virulent, LLO/sup> organisms and induced only one third of the amount of IFN-γ. Second, the ”bolus” injection of LLO would be expected to persist for a shorter period than the situation following the introduction of live, LLO-producing organisms that survive and continue to replicate for at least 7 days. The breakdown of the injected LLO would lose its effects on the innate response and thus on the immunomodulatory of the response. Third, LLO is cytolytic, and thus it is possible that directly injected LLO may induce the lysis of initially encountered monocytic cells recruited to the site of infection and thus interfere with the early response. It is possible to speculate that the observed potentiation may have resulted from the lowering of the infecting L. major dose through the lytic effects of LLO however our observations showed no such effect on L. major when exposed to LLO in vitro (data not shown).

In conclusion, our results demonstrate that LLO plays a major role in the observed immunopotentiation of the response of BALB/c mice against L. major. This effect, shown by the reduction in lesion size and parasite load, is mediated by the upregulation of IFN-γ production, and the subsequent control of parasite replication and dissemination.

Acknowledgements This work was supported by grants from the College of Higher Studies and the College of Medicine and Medical Sciences, Arabian Gulf University, Kingdom of Bahrain.
We extend our appreciation to Dr. Mirghani Osman for his assistance with the microscopic evaluation of tissue sections. Thanks are also extended to Dr. Mirghani Osman, Prof. Moiz Bakhiet and Prof. Giuseppe. Botta for their critical review of the manuscript. We acknowledge also the technical assistance of the late A. Gassim Al Tijani for the preparation of tissue sections.


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Correspondence: Khaled S. Tabbara, Department of Microbiology, Immunology and Infectious Diseases, College of Medicine and Medical Sciences, Arabian Gulf University, P.O. Box 22979, Manama, Kingdom of Bahrain. Phone: + (973) 239-733, fax: + (973) 230-730, e-mail tabara@agu.edu.bh





Copyright: © 2004 Polish Society of Experimental and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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