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. 2015 Oct 20;9(10):e0004178.
doi: 10.1371/journal.pntd.0004178. eCollection 2015.

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy

Ines Lakhal-Naouar  1 , Bonnie M Slike  2 , Naomi E Aronson  3 , Mary A Marovich  4
Affiliations

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy

Ines Lakhal-Naouar et al. PLoS Negl Trop Dis. .

Abstract

Background: The effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities.

Methods: We studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment.

Results: Healing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4+ T cell proliferation (hyporesponsiveness) seen with CD8+ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4+ T cells.

Conclusion: Healing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8+ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of lymphocyte populations by flow cytometry.
Data is presented from 30 subjects (17 in the SSG arm and 13 in TM arm represented in circles and triangles respectively) for which cells from all three time points were available. (A) Percentage of lymphocytes positive for surface expression of CD3 (T cells), CD19 (B cells), CD16/CD56 (CD3-: NK cells; CD3+: NKT-like cells). (B) Distribution of subpopulations of NK cells based on CD16 and CD56 expression. (C) T cell phenotype stratified by treatment arm. (D) Distribution of T cells into CD4+, CD8+ and CD4−CD8− (double negative, DN) populations pre- and post-treatment. (E-F) Identification of TCR expression within the T cell populations. Aggregate data from 12 study subjects compared to data from 7 healthy controls for (E) αβ and (F) γδ respectively. Bars represent medians. P values were derived using the Wilcoxon matched pairs test.
Fig 2
Fig 2. Lymphoproliferative response and cytokine production.
(A) Whole PBMC or CD8+T cell-depleted PBMC (CD8 depl PBMC) from 18 subjects (circles) and 16 subjects (triangles) treated respectively with SSG and TM at pre-treatment (black) and post-treatment (grey) stages were stimulated with SLA for 6 days followed by an 8 hour pulse with [3H]-thymidine. Lymphocyte stimulation index (LSI) was determined as fold-increase in mean cpm from triplicate wells over unstimulated wells. An LSI ≥ 5 (dotted line) is considered a positive response. (B) L. major antigen responses in whole PBMC stratified by treatment arm. (C-E) Cytokine production following stimulation with SLA. (C) IFN-γ, (D) TNF-α and (E) IL-10 production by total PBMC or CD8+ cell-depleted PBMC was quantified from supernatants sampled on day 6 of the lymphoproliferation assays. Bars represent medians. P values were derived using the Wilcoxon matched pairs test.
Fig 3
Fig 3. Identification of responding populations by CFDA-SE labeling and flow cytometry analysis.
Identification of proliferating lymphocytes based on expression of (A) CD4 and CD8 or (B) CD25. Circles represent SSG subjects and triangles represent TM subjects. Black and grey are for PRE and POST respectively. Bars represent medians.
Fig 4
Fig 4. Identification of activated and regulatory T cell populations by flow cytometry.
(A) Freshly isolated cells were stained for CD3, CD4, CD8 and CD25 for identification of activated T cells. Data obtained from 31 subjects (circles for SSG subjects and triangles for TM subjects) for which pre-treatment (black) and post-treatment (grey) cells were available. Bars represent medians. (B) Identification of Treg cells from thawed PBMC. Aggregate data from 20 donors for CD4+CD25high Foxp3+. P values derived using the Wilcoxon matched pairs test.
Fig 5
Fig 5. Characterization of the cytokine production capacity of responding T cells.
Multiparameter flow cytometry was used to determine (A) IFN-γ, IL-2, and TNF-α production in CD4+ T cells (B) the frequency of cells expressing each of the seven possible combinations of IFN-γ, IL-2, and TNF-α. Circles represent SSG subjects and triangles represent TM subjects. P values derived using the Mann-Whitney test for unpaired samples.
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Grants and funding

This work was supported by Department of Defense Global War on Terrorism funds and by a cooperative agreement [W81XWH-07-2-0067] between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. and the U.S. Department of Defense (DOD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.