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. 2020 Mar 2;221(6):973-982.
doi: 10.1093/infdis/jiz538.

Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis

Affiliations

Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis

Taís M Campos et al. J Infect Dis. .

Abstract

Background: Skin lesions from patients infected with Leishmania braziliensis has been associated with inflammation induced by cytotoxic CD8+ T cells. In addition, CD8+ T cell-mediated cytotoxicity has not been linked to parasite killing. Meanwhile, the cytotoxic role played by natural killer (NK) cells in cutaneous leishmaniasis (CL) remains poorly understood.

Methods: In this study, we observed higher frequencies of NK cells in the peripheral blood of CL patients compared with healthy subjects, and that NK cells expressed more interferon-γ, tumor necrosis factor (TNF), granzyme B, and perforin than CD8+ T cells.

Results: We also found that most of the cytotoxic activity in CL lesions was triggered by NK cells, and that the high levels of granzyme B produced in CL lesions was associated with larger lesion size. Furthermore, an in vitro blockade of granzyme B was observed to decrease TNF production.

Concclusions: Our data, taken together, suggest an important role by NK cells in inducing inflammation in CL, thereby contributing to disease immunopathology.

Keywords: CD8+ T cells; NK cells; cutaneous leishmaniasis; cytotoxic activity; granzyme B.

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Figures

Figure 1.
Figure 1.
Cytotoxic cell subsets in peripheral blood. (A) Representative plot showing cytotoxic cell subsets in peripheral blood based on expression of CD56, CD8, CD16 and CD3. (B) Frequency of cytotoxic cell subsets in peripheral blood of healthy subjects (HS) (N = 9) and cutaneous leishmaniasis (CL) patients (N = 12). Mann-Whitney test was used to compare cytotoxic cell subsets between HS and CL. *, P < .05.
Figure 2.
Figure 2.
Cytotoxic cells expressing interferon (IFN)-γ, tumor necrosis factor (TNF), granzyme B, and perforin in peripheral blood. Peripheral blood mononuclear cells were cultured in presence or absence of soluble Leishmania antigen (SLA) for 16 hours, then TNF and IFN-γ was intracellular stained in cytotoxic cell subsets. Frequency of granzyme B and perforin ex vivo expression was determined by intracellular staining in cytotoxic cell subsets. (A) Frequency of cytotoxic cell subsets expressing TNF in healthy subjects (HS) (N = 5) and cutaneous leishmaniasis (CL) patients (N = 4). (B) Frequency of cytotoxic cell subsets expressing IFN-γ in HS (N = 5) and CL patients (N = 11). (C) Frequency of granzyme B-producing cells in subsets of cytotoxic cells in HS (N = 5) and CL patients (N = 13). (D) Frequency of perforin-producing cells in subsets of cytotoxic cells in HS (N = 5) and CL patients (N = 13). The Wilcoxon test was used to compare results of different conditions in the same subset. The Mann-Whitney test was used to compare cytotoxic cell subsets between HS and CL. The Kruskal-Wallis test was used for comparisons of means between subsets of cytotoxic cells, Dunn’s posttest. *, P < .05; **, P < .005; ***, P < .0001.
Figure 3.
Figure 3.
NKG2D+CD4+ T cells produce interferon (IFN)-γ in patients with cutaneous leishmaniasis (CL). Peripheral blood mononuclear cells were cultured in presence or absence of soluble Leishmania antigen (SLA) for 16 hours, then granzyme B and IFN-γ were assessed by intracellular staining in NKG2D subsets. (A) Frequency of NKG2D in natural killer (NK) cells (healthy subjects [HS], N = 9; cutaneous leishmaniasis [CL], N = 19), CD8+ T cells (HS, N = 12; CL, N = 33), and CD4+ T cells (HS, N = 7; CL, N = 29) from peripheral blood of HS and CL patients. (B) Mean fluorescence intensity (MFI) of granzyme B in NKG2D+CD4+ T cells from CL patients (N = 9) with and without stimulus with SLA. (C) The MFI of IFN-γ in NKG2D+CD4+ T cells from CL patients (N = 29) with and without stimulus with SLA. Kruskal-Wallis test was used for comparisons of means between NK cells, CD8+ T cells, and CD4+ T cells expressing NKG2D. Wilcoxon test was used for comparisons of means before and after stimulus. *, P < .05; **, P < .005; ***, P < .0001.
Figure 4.
Figure 4.
Granzyme B production and natural killer (NK) cell degranulation in lesions of patients with cutaneous leishmaniasis (CL). (A) Biopsies of normal skin from healthy subjects (HS) (N = 4) and of lesions from CL patients (N = 9) were cultured for 12 hours without stimuli, and levels of granzyme B were determinated by enzyme-linked immunosorbent assay. (B) Flow cytometry gate strategy for determination of CD107a expression in NK cells and CD8+ T cells on lesions of CL patients. (C) Frequency of NK cells and CD8+ T cells in lesions of CL patients (N = 9). (D) CD107a expression on NK cells and CD8+ T cells on lesions of CL patients (N = 9). (E) Immunostaining for NK cells in tissue of lesion of CL patients. Slides were photographed (10 randomized fields from each section). Original magnification, ×ばつ20. Scale bar = 20 μm. Mann-Whitney test was used to compare different groups. **, P < .005; ***, P < .0001.
Figure 5.
Figure 5.
Granzyme B induces tumor necrosis factor (TNF) production and is associated with severity of disease. (A) Peripheral blood mononuclear cells from cutaneous leishmaniasis (CL) patients (N = 10) were stimulated with soluble Leishmania antigen (SLA) in the presence or absence of granzyme B inhibitor for 72 hours, and levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), and CXCL-10 were assessed by enzyme-linked immunosorbent assay. (B) Correlation analysis between granzyme B levels on supernatants of lesions, and lesion size, Leishmanin skin test, and days of illness of CL patients (N = 9). Statistical comparison was performed by Spearman’s rank correlation test according to nonparametric distribution of samples. Statistical significance, P < .05.

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