Low CXCL13 Expression, Splenic Lymphoid Tissue Atrophy and Germinal Center Disruption in Severe Canine Visceral Leishmaniasis

Visceral leishmaniasis is associated with atrophy and histological disorganization of splenic compartments. In this paper, we compared organized and disorganized splenic lymphoid tissue from dogs naturally infected with Leishmania infantum assessing the size of the white pulp compartments, the distribution of T, B and S100+ dendritic cells, using immunohistochemistry and morphometry and the expression of CCR7 and the cytokines, CXCL13, lymphotoxin (LT)-α, LT-β, CCL19, CCL21, TNF-α, IL-10, IFN-γ and TGF-β, using by real time RT-PCR. The lymphoid follicles and marginal zones were smaller (3.2 and 1.9 times, respectively; Mann-Whitney, P<0.02) in animals with disorganized splenic tissue in comparison to those with organized splenic lymphoid tissue. In spleens with disorganized lymphoid tissue, the numbers of T cells and S100+ dendritic cells were decreased in the follicles, and the numbers of B cells were reduced in both the follicles and marginal zones. CXCL13 mRNA expression was lower in animals with disorganized lymphoid tissue (0.5±0.4) compared to those with organized lymphoid tissue (2.7±2.9, both relative to 18S expression, P = 0.01). These changes in the spleen were associated with higher frequency of severe disease (7/12) in the animals with disorganized than in animals with organized (2/13, Chi-square, P = 0.01) splenic lymphoid tissue. The data presented herein suggest that natural infection with Leishmania infantum is associated with the impairment of follicular dendritic cells, CXCL13 expression, B cell migration and germinal center formation and associates these changes with severe clinical forms of visceral leishmaniasis. Furthermore the fact that this work uses dogs naturally infected with Leishmania infantum emphasizes the relevance of the data presented herein for the knowledge on the canine and human visceral leishmaniasis

Leukocyte populations in organized and disorganized spleens.

<p>Distribution of CD79α⁺ B and CD3⁺ T lymphocytes, S100⁺ dendritic cells and Ki-67⁺ proliferating cells in the spleens of dogs infected with <b><i>L. infantum</i></b> with and without disruption of splenic lymphoid tissue structure (figures <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>, <b>G</b> and <b>H</b>, bar = 70 µm; figures <b>E</b> and <b>F</b>, bar = 50 µm).</p

Estimation of the areas represented by the different compartments of the spleen in animals with organized or disorganized splenic histological architecture.

a<p>Organized/disorganized ratios;</p>b<p>Unpaired t test or Mann-Whitney test when recommended.</p>c<p>Proportion (%) of the splenic tissue represented by the specific compartment either red or white pulp in the histological sections;</p>d<p>Mean and sd of the compartment (PALS, follicle or marginal zone) area expressed in mµ².</p

Splenic white pulp compartments defined for morphometric estimative.

<p><b>A</b> – Dog spleen section showing the red pulp and the white pulp compartments; <b>B</b> – white pulp compartments estimated by morphometry. RP = red pulp, MZ = marginal zone, FL = follicle, P = PALS.</p

B cell, T cell, dendritic and proliferating cells number in organized and disorganized spleens.

<p>Estimation of the average number of CD79α⁺ B and CD3⁺ T lymphocytes, S100⁺ dendritic cells and Ki-67⁺ proliferating cells in cross sections of the splenic compartments of dogs infected with <i>L. infantum</i> with and without disruption of splenic lymphoid tissue structure. Box and whisker plots represent median, 25% and 75% percentiles and ranges. Y-axis scale is in Log₁₀. The number of animals used in each estimative is shown on the top of the up range bar. P values (Mann-Whitney test) only shown for statistically significant differences.</p

Cytokine expression in organized and disorganized spleens.

<p>The gene expression of cytokines and CCR7 in the splenic compartments of stray dogs uninfected with organized splenic white pulp (ORG-N), infected with <i>L. infantum</i> with (DES-P) and without (ORG-P) disruption of splenic lymphoid tissue structure. Horizontal lines represent the median of the estimates from RT-PCR, and the values are relative to 18S gene expression. Each dot corresponds to a different animal with 12 organized (12) or disorganized (9) splenic lymphoid tissue. P values (Kruskal-Wallis test) only shown for statistically significant differences.</p