Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10

Abstract

Background: The interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis.

Methods: IL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP.

Results: We showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals.

Conclusions: These data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.

Figure 1

IL-32 is detected in cutaneous and mucosal lesions of ATL patients. Fragments of cutaneous lesions (CL, n = 23, A) or mucosal lesions (ML, n = 9, B) and healthy tissues (skin, n = 8; mucosa, n = 7) were included in paraffin and sections were submitted to IHC for IL-32. After reaction, the expression of IL-32 was determined through positive cells, analysed under light microscopy. The data represent individuals and medians values. *p < 0.05. Panel C: photomicrography of skin control. Panel D: photomicrography of CL lesion positive for IL-32. Panel E: photomicrography of mucosa control. Panel F: photomicrography of ML lesion positive for IL-32.

Figure 2

Expression of IL-32 is detected in different cells. Fragments of cutaneous or mucosal lesions were included in paraffin and sections were submitted to IHC for IL-32. The arrows show positive cells: In panel A: mononuclear cell and giant cell; and Panel B: endothelial cells and giant cell. In Panel C: positive keratinocytes; and Panel D: several positive mononuclear cells. The expression of IL-32 was determined, under light microscopy, in epithelium and infiltrate of CL (E, n = 22) and ML (F, n = 9). The data represent individual and median values.

Figure 3

IL-32γ mRNA is detected in lesions of patients with cutaneous (CL) and mucosal (ML) leishmaniasis. Total mRNA was extracted from fragments of healthy tissues (C, skin n = 8; mucosa n = 7), cutaneous lesions (CL, n = 10) or mucosal lesions (ML, n = 7), and analysed by qPCR. Expression of isoforms α, β, γ and δ of IL-32 were analysed but only γIL-32 mRNA was detected. ND = Not detected. Results shown are individual values and medians. *p < 005.

Figure 4

Expression of TNF and IL-10 is increased in cutaneous and mucosal lesions of ATL patients. Fragments of cutaneous (CL, A and C), mucosal (ML, B and D), and healthy tissues (Control) were included in paraffin and sections were submitted to IHC for TNF (A and B) and IL-10 (C and D). After reaction, the expression of cytokines was determined through quantification of positive cells, under light microscopy (400×). Data represent individual and median values. TNF CL, n = 21; Control CL, n = 8. IL-10 CL, n = 23; Control CL, n = 8. TNF ML, n = 9; Control ML, n = 7. IL-10 ML and Control, n = 8. p < 0.05.

Figure 5

Increase of TNF mRNA and IL-10 mRNA in lesions of ATL patients: high levels of TNF mRNA in mucosal leishmaniasis. Total mRNA was extracted from fragments of healthy tissues (Control, skin n = 8; mucosa n = 8), cutaneous (CL, A and C, n = 10) or mucosal (ML, B and D, n = 7) lesions, and the expressions of TNF mRNA (A, B) and IL-10 (C, D) were analysed by qPCR. Results are showed as individual and median value. *p < 0.05.

Figure 6

L. (V.) braziliensis induces IL-32γ mRNA in human mononuclear cells. Mononuclear cells (3 × 10⁶/mL) from healthy blood donors were incubated with amastigote forms of L. (V.) braziliensis (3 × 10⁵/well) for 24 h. The IL-32γ mRNA was analysed by qPCR. Data represent individual values and medians (n = 21, *p < 0.05).