Pro-Cellular Exhaustion Markers are Associated with Splenic Microarchitecture Disorganization and Parasite Load in Dogs with Visceral Leishmaniasis

Abstract

In canine visceral leishmaniasis (CVL), splenic white pulp (SWP) disorganization has been associated with disease progression, reduced cytokine and chemokine expression and failure to control the parasite load. This profile is compatible with the cellular exhaustion previously shown in human visceral leishmaniasis. The present study aimed to evaluate the in situ expression of cellular exhaustion markers and their relation to clinical signs, SWP disorganization and parasite load. Forty dogs naturally infected by Leishmania infantum were grouped according to levels of SWP organization and parasite load. SWP disorganization was associated with reductions in the periarteriolar lymphatic sheath and lymphoid follicles/mm² and worsening of the disease. Apoptotic cells expressing CTLA-4⁺ increased in dogs with disorganized SWP and a high parasite load. In the same group, PD-L1 and LAG-3 gene expression were reduced. A higher number of CD21⁺TIM-3⁺ B cells was detected in disorganized spleens than in organized spleens. Apoptosis is involved in periarteriolar lymphatic sheath reduction and lymphoid follicle atrophy and is associated with CTLA-4⁺ cell reductions in the splenic tissue of dogs with visceral leishmaniasis (VL). Failure to control the parasite load was observed, suggesting that cell exhaustion followed by T and B cell apoptosis plays a role in the immunosuppression observed in CVL.

Figure 1

Identification of (A–C) CD4⁺, (D–F) CD8⁺, and (G–I) CD21⁺ cells by immunohistochemistry and quantitative analysis of the spleen of dogs naturally infected with L. infantum grouped according to the disorganization of the splenic white pulp and the parasite load: (B) percentage of CD4⁺ cells, (C) CD4⁺ cells/mm², (E) percentage of CD8⁺ cells, (F) CD8⁺ cells/mm², (H) percentage of CD21⁺ cells, and (I) CD21⁺ cells/mm². Mann-Whitney test: (B) *p = 0.019; (C) *p = 0.0036, **p = 0.0383. Kruskal-Wallis test: (B) p = 0,031; (C) p = 0.021.

Figure 2

Identification of (A–C) IFN-γ⁺, (D–F) IL-10⁺, and (G–I) Ki-67⁺ cells by immunohistochemistry and quantitative analysis of the spleen of dogs naturally infected with L. infantum grouped according to the clinical score: (B) percentage of IFN-γ⁺ cells, (C) IFN-γ cells/mm², (E) percentage of IL-10⁺ cells, (F) IL-10⁺ cells/mm², (H) percentage of Ki-67⁺ cells and (I) Ki-67⁺ cells/mm². Mann-Whitney test: (E) *p = 0.0409, **p = 0.0205. Kruskal-Wallis test: (E) p = 0.026.

Figure 3

Identification of (A–C) TIM-3⁺ and (D–F) CTLA-4⁺ cells by immunohistochemistry and quantitative analysis of exhausted cells in the spleen of dogs naturally infected with L. infantum grouped according to the disorganization of the splenic white pulp and the parasite load: (B) percentage of TIM-3⁺ cells, (C) TIM-3 cells/mm², (E) percentage of CTLA-4⁺ cells, and (F) CTLA-4⁺ cells/mm². Mann-Whitney test: (E) *p = 0.00160. Kruskal-Wallis test: (E) p = 0.021.

Figure 4

Gene expression of exhaustion markers in the spleen of dogs naturally infected with L. infantum. Ex vivo analysis by qPCR of the mRNA levels in the spleen of dogs classified according to the parasite load and splenic disorganization. Gene expression values were normalized to those of the constitutive genes HPRT and GAPDH. ANOVA: (B) p = 0.01; (D) p = 0.01. Tukey’s test (*): (B) p < 0.05; (D) p < 0.05.

Figure 5

Gene expression of exhaustion markers in the spleen of dogs naturally infected with L. infantum. Ex vivo analysis by qPCR of the mRNA levels in the spleen of dogs classified according to the clinical score. Gene expression values were normalized to those of the constitutive genes HPRT and GAPDH. ANOVA: (A) p < 0.022. (*) Mann-Whitney test: (B) p < 0.036.

Figure 6

Identification of CD21⁺ and TIM-3⁺ cells by immunofluorescence in the spleen of dogs naturally infected by L. infantum presenting organized (A–C) or disorganized (D–F) splenic white pulp. In (A,D) the presence of CD21⁺ cells/B lymphocytes (red PE) is shown. In (B,E) the presence of TIM-3⁺ cells (green FITC) is shown. In (C,F) overlapping images (red PE/green FITC/blue DAPI) are shown. Yellow arrows indicate CD21⁺ cells also expressing TIM-3⁺. Scale bar: 25 μm.

Figure 7

Identification of apoptotic cells by TUNEL staining of the spleen of dogs naturally infected by L. infantum presenting organized (A,B) or disorganized (C,D) splenic white pulp. Scale bar: 25 μm. (E) Quantitative analysis of apoptotic cells according to the disorganization of the splenic white pulp and the parasite load.

Figure 8

Identification of apoptotic CTLA-4⁺ cells by TUNEL staining of the spleen of dogs naturally infected by L. infantum presenting organized (A–C) or disorganized (D–F) splenic white pulp. Scale bar: 25 μm. Red arrows indicate isolated CTLA-4 expression. Green arrows indicate cells undergoing apoptosis without expressing CTLA-4. Yellow arrows indicate double labelling (CTLA-4⁺  + apoptosis). (G) Quantitative analysis of CTLA-4⁺ cells and apoptotic cells/mm² according to the disorganization of the splenic white pulp and the parasite load. ANOVA: p = 0.0263. Mann-Whitney test (*): (B) p = 0.0303.