Keratinocytes determine Th1 immunity during early experimental leishmaniasis

Abstract

Experimental leishmaniasis is an excellent model system for analyzing Th1/Th2 differentiation. Resistance to Leishmania (L.) major depends on the development of a L. major specific Th1 response, while Th2 differentiation results in susceptibility. There is growing evidence that the microenvironment of the early affected tissue delivers the initial triggers for Th-cell differentiation. To analyze this we studied differential gene expression in infected skin of resistant and susceptible mice 16h after parasite inoculation. Employing microarray technology, bioinformatics, laser-microdissection and in-situ-hybridization we found that the epidermis was the major source of immunomodulatory mediators. This epidermal gene induction was significantly stronger in resistant mice especially for several genes known to promote Th1 differentiation (IL-12, IL-1beta, osteopontin, IL-4) and for IL-6. Expression of these cytokines was temporally restricted to the crucial time of Th1/2 differentiation. Moreover, we revealed a stronger epidermal up-regulation of IL-6 in the epidermis of resistant mice. Accordingly, early local neutralization of IL-4 in resistant mice resulted in a Th2 switch and mice with a selective IL-6 deficiency in non-hematopoietic cells showed a Th2 switch and dramatic deterioration of disease. Thus, our data indicate for the first time that epidermal cytokine expression is a decisive factor in the generation of protective Th1 immunity and contributes to the outcome of infection with this important human pathogen.

Figure 1. Analysis of gene and protein expression in L. major infected skin.

A) RT-PCR Analysis of gene expression in 16 h L. major infected skin. The PCR data were normalized to GAPDH expression and mean N-fold regulation and SEM in comparison to PBS injected controls was calculated (n = 3). Grey bars: Regulation in BALB/c mice; Black bars: Regulation in C57BL/6 mice. * = P<0.05, ** = P<0.01, *** = P<0,001 for differences between C57BL/6 and BALB/c mice, Student's t-test. B) Protein secretion in L. major infected skin. Soluble CCL2, Opn, TNFα (16h after infection) and IL-6 (8h after infection) protein was measured in infected foots as described in Materials and Methods. Striped grey bars: Mean and SE in BALB/c mice; Black bars: Mean and SE C57BL/6 mice. The experiment was performed three times with similar results.

Figure 2. Analysis of gene in keratinocytes isolated from L. major infected skin.

Laser capture microdissection (left panel). Single keratinocytes were dissected from cryosections of infected and control skin as described in Materials and methods. A) Overview, cryosection (BALB/c mice 16 h after infection). B,C) Single keratinocytes of the stratum spinosum were selected, laser microdissected and pressure catapulted. RT-PCR Analysis of gene expression in microdissected keratinocytes from 16h L. major infected skin (right panel). The PCR data were normalized to GAPDH expression and N-fold regulation and SEM in comparison to PBS injected controls was calculated (n = 2). Grey bars: Regulation in BALB/c mice; Black bars: Regulation in C57BL/6 mice.

Figure 3. In-situ-hybridization and immunohistochemistry.

The cellular expression pattern of the indicated genes was analyzed in footpads of C57BL/6 mice infected 16 h with L. major by in-situ-hybridization (A) and immunohistochemistry (B). The arrow indicates Ym-1 positive cells in the infiltrate. Bars represent 50 µm; as = antisense RNA probe; s = sense RNA probe; Opn Ab = rat anti mouse monoclonal antibody against opn; rIgG = rat IgG.

Figure 4. RT-PCR Time course analysis of gene expression in 1h to 72h L. major infected skin.

The PCR data were normalized to GAPDH expression and mean N-fold regulation and SEM in comparison to PBS injected controls was calculated (n = 3) Grey bars: Regulation in BALB/c mice; Black bars: Regulation in C57BL/6 mice. * = P<0.05, ** = P<0.01, *** = P<0.001 for differences between C57BL/6 and BALB/c mice, Student's t-test.

Figure 5. Early anti-IL-4 treatment induces a Th2 switch.

IL-4, IL-13 and IFNγ secretion of CD4⁺ cells isolated one week after infection from draining lymph nodes of C57BL/6 mice which had been injected with 1 µg of neutralizing anti-IL-4 or irrelevant antibody into the infected footpad at the time of parasite inoculation and 4h later. Cells were incubated for 48 h with syngenic DC stimulated for 48h with soluble Leishmania antigen (sLmAg) (black bars) or with unstimulated syngenic DC (open bars). Cytokines in culture supernatants were assayed by cytometric bead assay (mean ± SE, n = 3) * = p<0.05, ** = p<0.01, Student's t-test.

Figure 6. Experimental leishmaniasis in wt→IL-6 −/− chimeric mice.

A) Footpad swelling (compared to the not infected contra lateral footpad) (mm) of infected wt→IL-6 −/− chimeric mice, control wt→wt mice and control IL-6 −/− →IL-6 −/− chimeric mice ** = p<0.01, Student's t-test. B) Limiting dilution assay (LDA) from footpads of wt→IL-6 −/− chimeric mice, control wt→wt mice and control IL-6 −/− →IL-6 −/− chimeric mice 9 weeks after infection (mean */− SE of log parasites) ** = p<0.01, Student's t-test. C) IFNγ and IL-13 secretion of CD4⁺ cells isolated from draining lymph nodes of wt→IL-6 −/− chimeric mice, control wt→wt mice and control IL-6 −/− →IL-6 −/− chimeric mice 9 weeks after infection. Cells were incubated for 48 h with syngenic dendritic cells stimulated for 48h with soluble Leishmania antigen (s LmAg) (black bars) or with unstimulated syngenic dendritic cells (without (w/o) Lm Ag, open bars). Cytokines in culture supernatants were assayed by cytometric bead assay (mean ± SE, n = 3) * = p<0.05, *** = p<0.001, Student's t-test.