Immunology and Cell Biology (2001) 79, 320–322 Research Article Susceptibility to Leishmania mexicana infection is due to the inability to produce IL-12 rather than lack of IL-12 responsiveness M I R I A M RO D R I G U E Z - S O S A , G I NA M M O N T E F O RT E a n d A B H AY R S ATO S K A R Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA Summary Almost all inbred mice are highly susceptible to parasites of the Leishmania mexicana complex that includes L. amazonensis and L. mexicana. Recent studies have reported that T cells from L. amazonensis-infected mice fail to respond to IL-12 due to impaired IL-12R expression. Here, we demonstrate that lymph node cells from L. mexicana-infected C57BL/6 and 129Sv/Ev mice respond efficiently to exogenous IL-12 in vitro and produce IFN-γ. Moreover, we also show that deletion of signal transducer and activator of transcription (STAT)4 gene in resistant STAT6–/– mice renders them susceptible to L. mexicana. These findings indicate that an inability to produce IL-12 rather than unresponsiveness to this cytokine is responsible for susceptibility to L. mexicana. Moreover, the data also demonstrate that the STAT4-mediated pathway is critical for the development of protective immunity against cutaneous leishmaniasis, regardless of the species of Leishmania and/or genetic background of the mice. Key words: Leishmania mexicana, STAT4, STAT6. Introduction American cutaneous leishmaniasis is caused by parasites of the Leishmania mexicana complex that includes L. amazonensis and L. mexicana.1 It is well documented that almost all inbred mice are susceptible to L. mexicana complex and develop large non-healing lesions following infection with these parasites.2,3 We have previously demonstrated that IL-4–/– as well as signal transducer and activator of transcription (STAT)6–/– mice produce high levels of IL-12, develop a Th1 response and control L. mexicana infection.4,5 These findings indicate that IL-4 and the STAT6-mediated signalling pathway mediate susceptibility to L. mexicana by suppressing IL-12 production and Th1 development. In contrast, other studies indicate that the inability to generate a Th1 response rather than the presence of IL-4 or a Th2 response may be responsible for susceptibility to L. amazonensis.6,7 Moreover, lack of Th1 in L. amazonensis-infected mice has been attributed to the inability of CD4+ T cells to respond to IL-12 due to the suppression of IL-12Rβ expression.7 Therefore, in this study, we determined whether lack of IL-12 production or inability to respond to this cytokine was responsible for impaired Th1 development and susceptibility to L. mexicana infection. Materials and Methods Animals Six to eight-week-old female C57BL/6 and 129Sv/Ev mice were purchased from Taconic Laboratories (Germantown, NY, USA). Correspondence: Dr AR Satoskar, Department of Microbiology, The Ohio State University, 484 W 12th Avenue, Columbus, OH 43210, USA. Email: [email protected] Received 1 December 2000; accepted 26 February 2001. STAT4/STAT6 gene-deficient C57BL/6 × 129Sv/Ev mice were generated by intercrossing STAT4–/– and STAT6–/– C57BL/6 × 129Sv/Ev mice that were kindly provided by Dr James Ihle (St. Jude’s Hospital, Memphis, TN, USA). These mice were bred and maintained in a facility at the Harvard School of Public Health according to the guidelines for animal research. Parasite and infection protocols For all experiments, mice were infected by injecting 5 × 106 L. mexicana (M379) amastigotes into shaven back rump. The disease progression was monitored by measuring lesion diameters weekly until week 10 postinfection. Antibody ELISA Mice were bled at different time points from tail snips following L. mexicana infection. Blood was centrifuged at 200 × g, and serum was collected and tested for L. mexicana antigen (LmAg)-specific Th1-associated IgG2a and Th2-associated IgG1 antibodies by ELISA as described previously.5 T-cell proliferation and cytokine assays At week 10 postinfection the mice were killed, their lymph nodes excised and T-cell proliferation assays were performed as described previously.5 Briefly, 3 × 105 lymph node cells were added to the wells of 96-well flat-bottomed tissue culture plates and stimulated with 20 µg/mL of LmAg prepared by repeated freezing and thawing of stationary phase promastigotes. Supernatants from these cultures were analysed for IL-4, IL-12 and IFN-γ production by ELISA.5 Results and Discussion It is widely accepted that IL-12 plays a critical in the development of Th1 response and protective immunity during Susceptibility to L. mexicana infection Figure 1 In vitro production of IFN-γ by L. mexicana antigen (LmAg)-stimulated lymph node cells from (a) C57BL/6 and (b) 129Sv/Ev mice following addition of recombinant murine IL-12. Five animals were used in each group. Data are expressed as means ± SEM and are representative of three experiments. Figure 2 Course of cutaneous L. mexicana infection in STAT4/STAT6+/+ (), STAT4/STAT6–/– () and STAT6–/– mice (). (a) Lesion growth was monitored by measuring lesion diameters following L. mexicana infection. Data are expressed as mean lesion diameter ± SE. At week 10 postinfection in vitro L. mexicana antigen (LmAg)-induced (b) IFN-γ and (c) IL-4 by the lymph node cells from STAT4/STAT6+/+ (wild type) and STAT4/STAT6–/– mice were measured by ELISA. Four to five animals were used in each group. 321 322 M Rodriguez-Sosa et al. cutaneous as well as visceral leishmaniasis.8,9 As demonstrated previously, C57BL/6 and 129Sv/Ev mice were highly susceptible to L. mexicana and developed large non-healing lesions following L. mexicana infection (data not shown). At week 10 postinfection, LmAg-stimulated lymph node cells from these mice failed to produce significant levels of IFN-γ, which was associated with basal levels of IL-12 (Fig. 1). However, addition of exogenous recombinant murine IL-12 to these cultures induced significant IFN-γ production by LmAg-stimulated lymph node cells from these inbred strains (Fig. 1). These results demonstrate that T cells, regardless of the genetic background of the mouse, can respond efficiently to IL-12 and produce IFN-γ during L. mexicana infection. Moreover, these results also suggest that lack of IL-12 production rather than responsiveness to this cytokine may be responsible for impaired IFN-γ production following L. mexicana infection. It is possible that IL-12-induced IFN-γ may be derived partly from other nonCD4+ T cells, such as NK cells. Nevertheless, these findings differ from those observed in previous studies demonstrating that the lack of IL-12 responsiveness resulting from impaired IL-12Rβ expression inhibits and mediates susceptibility to L. amazonensis.7 We previously found that genetically susceptible C57BL/ 6 × 129Sv/Ev mice lacking STAT6 gene produce significantly higher levels of IL-12, develop a Th1 response and control L. mexicana infection, suggesting that L. mexicana infection does not alter in vivo responsiveness of T cells to IL-12.5 If susceptibility to L. mexicana was due to the lack of IL-12 responsiveness, STAT6–/– mice would have failed to mount a Th1 response and remained susceptible to L. mexicana despite production of high levels of IL-12. Recent studies have demonstrated that CD4+ and CD8+ T cells differ in their regulation of IFN-γ.10 While STAT4-mediated IL-12R signalling is critical for IFN-γ production in CD4+ T cells via the TCR pathway and Th1 development, CD8+ T cells can produce IFN-γ independently of IL-12.10 Therefore, we hypothesized that deletion of STAT4 gene in STAT6–/– mice will impair Th1 development and render them susceptible to L. mexicana. As anticipated, in the present study we found that STAT4/STAT6–/– C57BL/6 × 129Sv/Ev mice developed rapidly progressive large non-healing lesions similar to STAT4/STAT6+/+ mice (Fig. 2a). In contrast, concomitantly infected STAT6–/– mice failed to develop lesions (Fig. 2a). At week 10 postinfection, LmAg-stimulated lymph node cells from STAT4/STAT6 mice produced significantly lower levels of IFN-γ and IL-4 than wild-type mice did (Fig. 2b,c). These observations demonstrate that in the absence of STAT4-mediated IL-12 signalling pathway STAT6–/– mice become susceptible to L. mexicana. These findings demonstrate that the IL-12R/STAT4-mediated pathway is functional and indispensable for Th1 development in STAT6–/– mice during L. mexicana infection. These findings support our previous observations in a Leishmania major model,11 and indicate that the STAT4-mediated pathway is critical for the development of protective immunity against cutaneous leishmaniasis regardless of the species of Leishmania and/or genetic background of the mice. In conclusion, our data indicate that unlike L. amazonensis, failure to develop an efficient Th1 response during L. mexicana infection is due to the lack of IL-12 production rather than the inability of T cells to respond to this cytokine. Moreover, the data also demonstrate that in the absence of STAT6, the STAT4-mediated IL-12 signalling pathway is indispensable for the development of protective immunity against L. mexicana. References 1 Peters W, Killick-Kendrick R. The Leishmaniases in Biology and Medicine. Vol. 1 and Vol. 2. London: Academic Press, 1987. 2 Alexander J, Satoskar AR, Russell DG. Leishmania species: models of intracellular parasitism. J. Cell Sci. 1999; 112: 2993–3002. 3 Blackwell JM. Protozoal infections. In: Wakelin DM, Blackwell JM (eds). Genetics of Resistance to Bacterial and Parasitic Infection. London: Taylor and Francis, 1988; 103–11. 4 Satoskar A, Bluethmann H, Alexander J. Disruption of the murine interleukin-4 gene inhibits disease progression during Leishmania mexicana infection but does not increase control of Leishmania donovani infection. Infect. Immun. 1995; 63: 4894–9. 5 Stamm LM, Raisenen-Sokolowski A, Okano M, Russell ME, David JR, Satoskar AR. Mice with STAT6-targeted gene disruption develop a Th1 response and control cutaneous leishmaniasis. J. Immunol. 1998; 161: 6180–8. 6 Afonso LCC, Scott P. Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis. Infect. Immun. 1993; 61: 2952–9. 7 Jones DE, Baxbaum LU, Scott P. IL-4-independent inhibition of IL-12-responsiveness during Leishmania amazonensis infection. J. Immunol. 2000; 165: 364–72. 8 Satoskar AR, Rodig S, Telford SR, Satoskar AA, Ghosh SK, von Lichtenberg F, David JR. IL-12 gene deficient C57BL/6 mice are susceptible to L. donovani but have diminished hepatic immunopathology. Eur. J. Immunol. 2000; 30: 834–9. 9 Scharton-Kersten T, Afonso LC, Wysocka M, Trinchieri G, Scott P. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol. 1995; 154: 5320. 10 Carter LL, Murphy KM. Lineage-specific requirement for signal transducer and activator of transcription (Stat) 4 in interferon gamma production from CD4(+) versus CD8(+) T cells. J. Exp. Med. 1999; 189: 1355–60. 11 Stamm LM, Satoskar AA, Ghosh SK, David JR, Satoskar AR. STAT4 mediated IL-12 signaling pathway is critical for the development of protective immunity in cutaneous leishmaniasis. Eur. J. Immunol. 1999; 29: 2524–9.
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