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We postulated that CCR2-driven activation of the transcription factor NF-κB plays a critical role in dendritic cell (DC) maturation (e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-κB were required for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr2−/− bone marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2 plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of DC populations such as LCs. First, compared with WT, Ccr2−/− lymph node cells, splenocytes, BMDCs, and LCs produced lower levels of IL-12p70 following stimulation with LPS/IFN-γ or L. major. Second, a reduced number of LCs carried L. major from the skin to the draining lymph nodes in Ccr2−/− mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed the susceptibility to L. major infection in Ccr2−/− mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our data point to an important role for CCR2-driven activation of NF-κB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens.

Active human visceral leishmaniasis (VL) is characterized by a progressive increase in visceral parasite burden, cachexia, massive splenomegaly, and hypergammaglobulinemia. In contrast, mice infected with Leishmania donovani, the most commonly studied model of VL, do not develop overt, progressive disease. Furthermore, mice control Leishmania infection through the generation of NO, an effector mechanism that does not have a clear role in human macrophage antimicrobial function. Remarkably, infection of the Syrian hamster (Mesocricetus auratus) with L. donovani reproduced the clinicopathological features of human VL, and investigation into the mechanisms of disease in the hamster revealed striking differences from the murine model. Uncontrolled parasite replication in the hamster liver, spleen, and bone marrow occurred despite a strong Th1-like cytokine (IL-2, IFN-γ, and TNF/lymphotoxin) response in these organs, suggesting impairment of macrophage effector function. Indeed, throughout the course of infection, inducible NO synthase (iNOS, NOS2) mRNA or enzyme activity in liver or spleen tissue was not detected. In contrast, NOS2 mRNA and enzyme activity was readily detected in the spleens of infected mice. The impaired hamster NOS2 expression could not be explained by an absence of the NOS2 gene, overproduction of IL-4, defective TNF/lymphotoxin production (a potent second signal for NOS2 induction), or early dominant production of the deactivating cytokines IL-10 and TGF-β. Thus, although a Th1-like cytokine response was prominent, the major antileishmanial effector mechanism that is responsible for control of infection in mice was absent throughout the course of progressive VL in the hamster.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1α (MIP-1α), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1α, or CCR2 had reduced IFN-γ responses following ligation of the TCR. Reduced IFN-γ responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5−/− and CCR2−/− mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-γ responses. However, this reduced IFN-γ response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-γ response was evident in CCR5−/− and MIP-1α−/− mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1α, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1α, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-γ, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1–3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-γ response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.