Genetics of Host Response to Leishmania tropica in Mice – Different Control of Skin Pathology, Chemokine Reaction, and Invasion into Spleen and Liver

Several hundred million people are exposed to the risk of leishmaniasis, a disease caused by intracellular protozoan parasites of several Leishmania species and transmitted by phlebotomine sand flies. In humans, L. tropica causes cutaneous form of leishmaniasis with painful and long-persisting lesions in the site of the insect bite, but the parasites can also penetrate to internal organs. The relationship between the host genes and development of the disease was demonstrated for numerous infectious diseases. However, the search for susceptibility genes in the human population could be a difficult task. In such cases, animal models may help to discover the role of different genes in interactions between the parasite and the host. Unfortunately, the literature contains only a few publications about the use of animals for L. tropica studies. Here, we report an animal model suitable for genetic, pathological and drug studies in L. tropica infection. We show how the host genotype influences different disease symptoms: skin lesions, parasite dissemination to the lymph nodes, spleen and liver, and increase of levels of chemokines CCL2, CCL3 and CCL5 in serum

Distinct genetic control of parasite elimination, dissemination, and disease after Leishmania major infection

Elimination of pathogens is the basis of host resistance to infections; however, relationship between persisting pathogens and disease has not been clarified. Leishmania major infection in mice is an important model of host–pathogen relationship. Infected BALB/c mice exhibit high parasite numbers in lymph nodes and spleens, and a chronic disease with skin lesions, splenomegaly, and hepatomegaly, increased serum IgE levels and cytokine imbalance. Although numerous gene loci affecting these disease symptoms have been reported, genes controlling parasites’ elimination or dissemination have never been mapped. We therefore compared genetics of the clinical and immunologic symptomatology with parasite load in (BALB/c × CcS-11) F2 hybrids and mapped five loci, two of which control parasite elimination or dissemination. Lmr5 influences parasite loads in spleens (and skin lesions, splenomegaly, and serum IgE, IL-4, and IFNγ levels), and Lmr20 determines parasite numbers in draining lymph nodes (and serum levels of IgE and IFNγ), but no skin or visceral pathology. Three additional loci do not affect parasite numbers but influence significantly the disease phenotype—Lmr21: skin lesions and IFNγ levels, Lmr22: IL-4 levels, Lmr23: IFNγ levels, indicating that development of L. major-caused disease includes critical regulations additional to control of parasite spread

Skin lesion caused by <i>Leishmania tropica</i> in female of CcS-16 strain at week 43 after infection.

<p>Skin lesion caused by <i>Leishmania tropica</i> in female of CcS-16 strain at week 43 after infection.</p

Mapping the Genes for Susceptibility and Response to <i>Leishmania tropica</i> in Mouse

<div><p>Background</p><p><i>L. tropica</i> can cause both cutaneous and visceral leishmaniasis in humans. Although the <i>L. tropica</i>-induced cutaneous disease has been long known, its potential to visceralize in humans was recognized only recently. As nothing is known about the genetics of host responses to this infection and their clinical impact, we developed an informative animal model. We described previously that the recombinant congenic strain CcS-16 carrying 12.5% genes from the resistant parental strain STS/A and 87.5% genes from the susceptible strain BALB/c is more susceptible to <i>L. tropica</i> than BALB/c. We used these strains to map and functionally characterize the gene-loci regulating the immune responses and pathology.</p><p>Methods</p><p>We analyzed genetics of response to <i>L. tropica</i> in infected F₂ hybrids between BALB/c×CcS-16. CcS-16 strain carries STS-derived segments on nine chromosomes. We genotyped these segments in the F₂ hybrid mice and tested their linkage with pathological changes and systemic immune responses.</p><p>Principal Findings</p><p>We mapped 8 <i>Ltr</i> (<i>Leishmania tropica</i> response) loci. Four loci (<i>Ltr2</i>, <i>Ltr3</i>, <i>Ltr6</i> and <i>Ltr8</i>) exhibit independent responses to <i>L. tropica</i>, while <i>Ltr1</i>, <i>Ltr4</i>, <i>Ltr5</i> and <i>Ltr7</i> were detected only in gene-gene interactions with other <i>Ltr</i> loci. <i>Ltr3</i> exhibits the recently discovered phenomenon of transgenerational parental effect on parasite numbers in spleen. The most precise mapping (4.07 Mb) was achieved for <i>Ltr1</i> (chr.2), which controls parasite numbers in lymph nodes. Five <i>Ltr</i> loci co-localize with loci controlling susceptibility to <i>L. major</i>, three are likely <i>L. tropica</i> specific. Individual <i>Ltr</i> loci affect different subsets of responses, exhibit organ specific effects and a separate control of parasite load and organ pathology.</p><p>Conclusion</p><p>We present the first identification of genetic loci controlling susceptibility to <i>L. tropica</i>. The different combinations of alleles controlling various symptoms of the disease likely co-determine different manifestations of disease induced by the same pathogen in individual mice.</p></div

Sex differences in lesion size (week 8) after <i>L. major</i> infection.

<p>Analysis of sex influence on lesion development was performed using General Linear Models Univariate ANOVA with experiment as a random and age as a fixed parameter. We have observed influence of experiment (<i>P</i> range 0.006–0.000019), whereas influence of age was not significant (<i>P</i>>0.17).</p

Number of parasites cultivated from lymph nodes of mouse strains 21 and 43 weeks after infection.

<p>Mice were killed at week 21 (A) and week 43 (B) after infection. Asterisks show strains that exhibited parasite load significantly different from BALB/c. Brackets indicate strains with differences between males and females. Data are presented as mean ± SD.</p

Interaction between loci controlling parasite burden in lymph nodes and liver 43 weeks after infection.

<p>Means, SE and <i>P</i> values for concentration of parasite DNA (ng/µl) in isolates from lymph nodes and liver were computed by analysis of variance. The following transformations were used to obtain normal distribution: natural logarithm of (value×100). Hepatomegaly (liver-to-body weight ratio×1000) was normalized by raising values to the power of 0.0125. The numbers in bold give the average non-transformed values. Only <i>P</i> values significant after correction for genome-wide significance are given. Number of tested mice is shown in brackets. C and S indicate the presence of BALB/c and STS allele, respectively.</p

Interactions among loci that control response to <i>L. tropica</i>.

<p>Phenotypes controlled by each locus are shown at its symbol in different colors. The colored lines connecting the loci indicate interactions controlling the specific phenotypes.</p

Kinetics of skin lesion development of CcS/Dem strains after infection with <i>L. tropica</i>.

<p>Individual strains are marked with different colors. The columns show median values of lesion size in females (A) and males (B). Figure summarizes data from two independent experiments.</p

Comparison of kinetics of serum level of CCL3 and CCL5 in females and males of strains CcS-16 and BALB/c.

<p>Kinetics of CCL3 levels (median values) in serum of BALB/c (A) and CcS-16 (B) and serum levels of CCL5 (median values) in BALB/c (C) and CcS-16 (D) mice is shown. Figure summarizes data from two independent experiments (21 and 43 weeks of infection).</p