Enhancement of experimental cutaneous leishmaniasis by Leishmania molecules is dependent on interleukin-4, serine protease/esterase activity, and parasite and host genetic backgrounds

Abstract

Most inbred strains of mice, like the BALB/c strain, are susceptible to Leishmania amazonensis infections and resistant to Leishmania braziliensis infections. This parasite-related difference could result from the activity of an L. amazonensis-specific virulence factor. In agreement with this hypothesis, it is shown here that the intravenous injection of BALB/c mice with L. amazonensis amastigote extract (LaE) but not the L. braziliensis extract confers susceptibility to L. braziliensis infection. This effect was associated with high circulating levels of IgG1 anti-L. amazonensis antibodies and with an increase in interleukin-4 (IL-4) production and a decrease in gamma interferon production by draining lymph node cells. Moreover, the effect was absent in IL-4-knockout mice. The biological activity in the LaE was not mediated by amphiphilic molecules and was inhibited by pretreatment of the extract with irreversible serine protease inhibitors. These findings indicate that the LaE contains a virulence-related factor that (i) enhances the Leishmania infection by promoting Th2-type immune responses, (ii) is not one of the immunomodulatory Leishmania molecules described so far, and (iii) is either a serine protease or has an effect that depends on that protease activity. In addition to being Leishmania species specific, the infection-enhancing activity was also shown to depend on the host genetic makeup, as LaE injections did not affect the susceptibility of C57BL/6 mice to L. braziliensis infection. The identification of Leishmania molecules with infection-enhancing activity could be important for the development of a vaccine, since the up- or downmodulation of the immune response against a virulence factor could well contribute to controlling the infection.

FIG. 1.

Injections of Leishmania amazonensis extract or its hydrophilic and amphiphilic fractions but not Leishmania braziliensis extract increased lesion sizes and parasite burdens of BALB/c mice infected in the footpad with 10⁷ Leishmania braziliensis stationary-phase promastigotes. Each mouse received four biweekly intravenous injections of the extracts or their fractions, starting 1 week before the infection, as detailed in the Materials and Methods. (A) Mice were infected with L. amazonensis (La) or with L. braziliensis (Lb). The L. braziliensis-infected mice were treated with LaE, L. braziliensis extract (LbE), or saline. Data are representative of those from five independent experiments. (B) Parasite burden, assessed by limiting dilution 6 weeks after infection with L. braziliensis, in the footpads of mice treated with LaE or saline in the experiment that produced the results depicted in panel A. Each circle represents the result obtained from an individual animal, the horizontal continuous lines indicate the medians of the results, and the horizontal broken line indicates the upper limit of the 75th percentile of the saline-treated group. (C) Mice were infected with L. braziliensis and injected with L. amazonensis axenic amastigote extract, L. amazonensis stationary-phase promastigote extract, or saline. The result is representative of those from two experiments. (D) Mice were infected with L. braziliensis and treated with hydrophilic or amphiphilic fractions of L. amazonensis extract or with the vehicle, i.e., Triton X-114-treated saline. In panels A, C, and D, each point represents the median lesion size from five mice and the vertical bars correspond to the interquartile interval. *, P < 0.05 (in relation to the saline group).

FIG. 2.

Injections of LaE do not affect the lesion sizes or parasite burdens in C57BL/6 mice infected in the footpad with 10⁷ Leishmania braziliensis stationary-phase promastigotes. Each BALB/c or C57BL/6 mouse received four biweekly intravenous injections of LaE or saline, starting 1 week before the infection, as detailed in the Materials and Methods. Each circle represents the result obtained from an individual animal. (A) Lesion sizes. The curves are defined by the median values obtained in groups of five or six mice. (B) Parasite burden, as assessed by limiting dilution 5 weeks after infection with L. braziliensis, in the footpads of the mice of the experiment that produced the results depicted in panel A. The horizontal lines indicate the medians of the results. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

FIG. 3.

Protease inhibitors reduce the infection-enhancing activity of L. amazonensis amastigote extract in BALB/c mice infected in the footpad with 10⁷ stationary-phase L. braziliensis promastigotes. Mice were treated with four biweekly injections of saline, L. amazonensis amastigote extract, or L. amazonensis amastigote extract supplemented with protease inhibitors, starting 1 week before the infection, as detailed in the Materials and Methods. (A) Lesion sizes in the footpads of mice treated with LaE, saline, or LaE supplemented with a mixture of serine and/or cysteine protease inhibitors (TPCK, TLCK, NPGB, and PMSF; LaE + SP/CP inhibitors). The data are representative of those from five independent experiments. (B) Parasite burden, as assessed by limiting dilution 5 weeks after infection with L. braziliensis, in the experiment that produced the results depicted in panel A. The horizontal broken line indicates the upper limit of the 75th percentile of the saline-treated group. (C) Lesion sizes in the footpads of mice treated with LaE, saline, LaE supplemented with a mixture of two serine protease inhibitors (TPCK and TLCK; LaE + SP inhibitors), LaE supplemented with a cysteine protease inhibitor (iodoacetamide; LaE + CP inhibitor), and LaE supplemented with a mixture of TPCK, TLCK, and iodoacetamide (LaE + SP & CP inhibitors). (D) Parasite burden 5 weeks after infection with L. braziliensis in the experiment that produced the results depicted in panel C. In panels A and C, the curves are defined by the median values obtained in groups of five to nine mice; in panels B and D, the horizontal lines indicate the median values of the group results. *, P < 0.01; **, P < 0.05; ns, nonsignificant.

FIG. 4.

Levels of anti-L. amazonensis extract antibodies in the sera of BALB/c mice infected in the footpad with 10⁷ stationary-phase L. braziliensis promastigotes. IgG1 (A) and IgG2a (B) antibodies were measured in sera, prepared from blood collected 6 weeks after the infection, of mice that had been treated with four biweekly injections of saline, LaE, or LaE supplemented with a mixture of serine/cysteine protease inhibitors (LaE + inhibitors), starting 1 week before the infection, as detailed in the Materials and Methods. The horizontal lines indicate the median value of each group of five animals. *, P < 0.001.

FIG. 5.

Amounts of cytokines produced by draining lymph nodes cells of BALB/c mice infected in the footpad with 10⁷ stationary-phase L. braziliensis promastigotes. The mice were treated with four biweekly injections of saline or LaE, starting 1 week before the infection. The lymph node cells, collected 1 week after the last saline or extract injection (i.e., 5 weeks after the infection), were in vitro stimulated with anti-CD3, and their supernatants were assayed for IL-4 (A) and IFN-γ (B), as detailed in the Materials and Methods. The horizontal lines indicate the median value of each group of five or six animals. *, P < 0.05.

FIG. 6.

Injections of L. amazonensis extract do not affect lesion sizes or parasite burden of IL-4^−/− BALB/c mice infected in the footpad with 10⁷ L. braziliensis stationary-phase promastigotes, determined 6 weeks after infection. (A) Lesion sizes; (B) parasite burdens. The mice received four biweekly intravenous injections of LaE or saline, starting 1 week before the infection, as detailed in the Materials and Methods. The horizontal lines indicate the median value of each group of 10 animals, and the data are representative of those from two independent experiments. *, P < 0.05; ns, nonsignificant.