Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum-naturally infected dogs

Abstract

Canine Visceral Leishmaniasis (CVL) shares many aspects with the human disease and dogs are considered the main urban reservoir of L. infantum in zoonotic VL. Infected dogs develop progressive disease with a large clinical spectrum. A complex balance between the parasite and the genetic/immunological background of the host are decisive for infection evolution and clinical outcome. This study comprised 92 Leishmania infected mongrel dogs of various ages from Mato Grosso, Brazil. Spleen samples were collected for determining parasite load, humoral response, cytokine mRNA expression and histopathology alterations. By real-time PCR for the ssrRNA Leishmania gene, two groups were defined; a low (lowP, n = 46) and a high parasite load groups (highP, n = 42). When comparing these groups, results show variable individual humoral immune response with higher specific IgG production in infected animals but with a notable difference in CVL rapid test optical densities (DPP) between highP and lowP groups. Splenic architecture disruption was characterized by disorganization of white pulp, more evident in animals with high parasitism. All cytokine transcripts in spleen were less expressed in highP than lowP groups with a large heterogeneous variation in response. Individual correlation analysis between cytokine expression and parasite load revealed a negative correlation for both pro-inflammatory cytokines: IFNγ, IL-12, IL-6; and anti-inflammatory cytokines: IL-10 and TGFβ. TNF showed the best negative correlation (r2 = 0.231; p<0.001). Herein we describe impairment on mRNA cytokine expression in leishmania infected dogs with high parasite load associated with a structural modification in the splenic lymphoid micro-architecture. We also discuss the possible mechanism responsible for the uncontrolled parasite growth and clinical outcome.

Fig 1. Quantification of parasite load in spleen samples from dogs naturally infected with Leishmania infantum.

Quantification was carried out using real time PCR with primers specific for a Leishmania sp. DNA sequence of the small subunit ribosomal RNA (ssrRNA). The canine HPRT gene was used in order to normalize initial concentrations of DNA in each sample. (A) Histogram and normal density lines of L. infantum infected animals classified into low (n = 46), red line, or high (n = 42), green line, parasite load by the fitting of an optimum number of mixtures of normal distributions. (B) Minimum and maximum values of parasite per 10⁶ canine cells. Each sample was quantified in duplicate for each target. a,b Statistically significant differences (p < 0.0001, Unpaired T-test).

Fig 2. Most animals with high parasite load present clinical signs.

Clinical signs in Leishmania infantum infected animals classified into low (n = 46) or high (n = 42) parasite DNA load measured by real-time PCR for the Leishmania ssrRNA gene in spleen.

Fig 3. Leishmania infantum naturally infected dogs present increased levels of immunoglobulins.

Leishmania-specific total IgG (A and C) and total IgM (B) levels in sera from noninfected dogs (control, n = 15) and from naturally infected dogs with either low (lowP, n = 46) or high (highP, n = 42) parasite load in spleen. Optical density of serum samples were determined by ELISA (A and B) using crude protein derived from L. infantum promastigotes IOC/L3128 (MCAN/BR/2009/BOB-FÍGADO). Reflectance values were determined by the rapid immunocromatographic test (Dual Path Platform, DPP, BioManguinhos). Horizontal bars indicate mean values. The dashed line indicates the cut-off, 0.549 for IgG and 0.645 for IgM. (***) p < 0.001; (**) p < 0.0002 indicate statistically significant differences, Mann Whitney test.

Fig 4. Pro-inflammatory and regulatory/anti-inflammatory cytokine mRNA expression correlates with low parasite load.

Ex-vivo analyses of relative mRNA levels for indicated genes in the splenic compartments of mongrel dogs infected with Leishmania infantum were correlated with individual log of spleen parasite load detected by pPCR for the Leishmania sp ssrRNA gene. Gene expression levels of each tested cytokine were normalized using HPRT and RP32 expression. For parasite load values, canine HPRT primers were used in order to normalize initial concentrations of DNA in each sample. P value < 0.05 indicates significant correlation, Pearson correlation test.

Fig 5. Splenic mRNA cytokines are down regulated in Leishmania infantum infected dogs with higher parasite loads.

Ex-vivo analyses of relative mRNA levels for indicated genes in the splenic compartments of mongrel dogs infected with L. infantum and classified as low (lowP) or high (highP) parasite load. Gene expression levels of each tested cytokine were normalized using HPRT and RP32 expression. Error bars indicate the standard error of mean for each group. Significant differences are indicated by, p < 0.05 and **, p < 0.01 (nonparametric T-test with 1,000 permutations).

Fig 6. Dogs with high parasite load show moderately to extensively disorganized white pulp.

Degree of white pulp organization by histopathology in Leishmania infantum infected animals classified into low (n = 33) or high (n = 26) parasite DNA load measured by real-time PCR for ssrRNA Leishmania gene in spleen.

Fig 7. Morphological changes in the spleen of dogs naturally infected by Leishmania infantum.

(A) Organized splenic white pulp compartments with well-formed follicle; (B) Moderately disorganized splenic white pulp when the white pulp was evident, however, its regions were poorly individualized or indistinct; (C) Disorganized splenic white pulp without compartment distinction; (D, E and F) Intracelullar amastigotes (arrows). HE, Scale bar 20 μm (A, B and C); 10 μm (D, E and F). WP—white pulp; RP—red pulp; BCA—B cell area; PALS—periarterial lymphoid sheat; CA—central artery.