SummaryIntracellular pathogens modulate host cell function to promote their survival. However, in vitro infection studies do not account for the impact of host-derived inflammatory signals. Examining the response of liver-resident macrophages (Kupffer cells) in mice infected with the parasite Leishmania donovani, we identified a transcriptomic network operating in uninfected Kupffer cells exposed to inflammation but absent from Kupffer cells from the same animal that contained intracellular Leishmania. To test the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival, we pharmacologically perturbed the activity of retinoid X receptor alpha (RXRα), a key hub within this network, and showed that this intervention enhanced the innate resistance of Kupffer cells to Leishmania infection. Our results illustrate a broadly applicable strategy for understanding the host response to infection in vivo and identify Rxra as the hub of a gene network controlling antileishmanial resistance.

Leishmania spp. are intracellular parasites that cause lesions in the skin, mucosa, and viscera. We have previously shown that Leishmania infection reduces mononuclear phagocyte adhesion to inflamed connective tissue. In this study, we examined the role of adhesion molecules and chemokines in this process. Infection rate (r ‫؍‬ ؊0.826, P ‫؍‬ 0.003) and parasite burden (r ‫؍‬ ؊0.917, P ‫؍‬ 0.028) negatively correlated to mouse phagocyte adhesion. The decrease (58.7 to 75.0% inhibition, P ‫؍‬ 0.005) in phagocyte adhesion to connective tissue, induced by Leishmania, occurred as early as 2 h after infection and was maintained for at least 24 h. Interestingly, impairment of cell adhesion was sustained by phagocyte infection, since it was not observed following phagocytosis of killed parasites (cell adhesion varied from 15.2% below to 24.0% above control levels, P > 0.05). In addition, Leishmania infection diminished cell adhesion to fibronectin (54.1 to 96.2%, P < 0.01), collagen (15.7 to 83.7%, P < 0.05), and laminin (59.1 to 82.2%, P < 0.05). The CD11b hi subpopulation was highly infected (49.6 to 97.3%). Calcium and Mg 2؉ replacement by Mn 2؉ , a treatment that is known to induce integrins to a high state of affinity for their receptors, reverted the inhibition in adhesion caused by Leishmania. This reversion was completely blocked by anti-VLA4 antibodies. Furthermore, expression of CCR4 and CCR5, two chemokine receptors implicated in cell adhesion, was found to be downregulated 16 h after infection (2.8 to 4.1 times and 1.9 to 2.8 times, respectively). Together, these results suggest that mechanisms regulating integrin function are implicated in the change of macrophage adhesion in leishmaniasis.

In this work, we have developed an adhesion assay to study interactions between mononuclear phagocytes and connective tissue in vitro and show its potential use to study diseases caused by intracellular microorganisms. The assay reproduces most of the characteristics of macrophage adhesion to connective tissue in vivo, such as: preferential adhesion to inflamed connective tissue, divalent cation and integrin dependence, and up-regulation upon cell activation. The phagocyte adhesion to connective tissue was inhibited by infection with Leishmania (58+/-22%, p < 0.05) and was not affected by infection with Mycobacterium or by endocytosis of latex beads. Manganese partially reverted the loss in adherence produced by Leishmania infection, indicating that the mechanisms regulating the function of integrins are affected by cell infection with Leishmania. This assay might be a useful tool for the study of the mechanisms by which mononuclear phagocytes play a role in the immune-inflammatory response and in the development of lesions.

BackgroundIn vitro studies show that Leishmania infection decreases the adhesion of inflammatory phagocytes to connective tissue by a mechanism dependent on the modulation of integrin function. However, we know little about the influence of this reduction in leukocyte adhesion on parasite dissemination from the infection site.MethodsIn this work, we used a model of chronic peritonitis induced by thioglycollate to study the effect of L. amazonensis infection on the ability of inflammatory phagocyte populations to migrate from an inflammatory site to the draining lymph node. Uninfected or Leishmania-infected thioglycollate-elicited peritoneal exudate cells were transferred from C57BL/6 to BALB/c mice or from Ly5.1+ to Ly5.1- mice. The transferred cells were injected into the peritoneal cavity and tracked to the draining lymph node.ResultsMigrating cells corresponded to approximately 1% of the injected leukocytes. The proportion of migrating CD11b+CD11c+ (myeloid dendritic cell) was lower after incubation with Leishmania (1.3 to 2.6 times lower in the experiments using C57BL/6 to BALB/c animals and 2.7 to 3.4 times lower in the experiments using Ly5.1+ to Ly5.1- animals) than after leukocyte incubation with medium alone (P < 0.01). There was no consistent decrease in the migration of CD11b+F4/80+ (macrophage) or SSChi GR-1+ (neutrophil) populations.ConclusionsCoincubation with Leishmania changes the migratory pattern of dendritic cells in vivo. Such changes in dendritic cell migration may be associated with immunological events that maintain inflammation at the sites of infection.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-450) contains supplementary material, which is available to authorized users.