Leishmania majorInfection Activates NF-κB and Interferon Regulatory Factors 1 and 8 in Human Dendritic Cells

Abstract

The salient feature of dendritic cells (DC) is the initiation of appropriate adaptive immune responses by discriminating between pathogens. Using a prototypic model of intracellular infection, we previously showed that Leishmania major parasites prime human DC for efficient interleukin-12 (IL-12) secretion. L. major infection is associated with self-limiting cutaneous disease and powerful immunity. In stark contrast, the causative agent of visceral leishmaniasis, Leishmania donovani, does not prime human DC for IL-12 production. Here, we report that DC priming by L. major infection results in the early activation of NF-B transcription factors and the up-regulation and nuclear translocation of interferon regulatory factor 1 (IRF-1) and IRF-8. The inhibition of NF-B activation by the pretreatment of DC with caffeic acid phenethyl ester blocks L. major-induced IRF-1 and IRF-8 activation and IL-12 expression. We further demonstrate that IRF-1 and IRF-8 obtained from L. major-infected human DC specifically bind to their consensus binding sites on the IL-12p35 promoter, indicating that L. major infection either directly stimulates a signaling cascade or induces an autocrine pathway that activates IRF-1 and IRF-8, ultimately resulting in IL-12 transcription.Leishmania major is the causative agent of cutaneous leishmaniasis, which is characterized by the development of lesions at sand fly bite sites. These cutaneous lesions ulcerate, resolve, and ultimately stimulate powerful immunity against the disease. Robust induction of this immunity is the basis of leishmanization, an effective vaccination procedure in which the inoculation of live L. major has been used with great success (12); safety concerns, however, have led to the abandonment of such vaccination (17). In contrast to L. major, Leishmania donovani causes visceral leishmaniasis, a severe systemic illness that is often fatal if untreated.Healing of cutaneous leishmaniasis has been attributed to the development of a strong Th1 immune response in the vertebrate host. Interleukin-12 (IL-12) is up-regulated in L. major-infected human dendritic cells (DC), whereas visceral leishmaniasis agents do not induce IL-12 production (28). Those previous studies revealed that L. donovani does not actively inhibit IL-12 production; rather, L. major primes human DC for IL-12 production. This strong induction of IL-12 by L. major-infected DC sets the stage for a strong and robust Th1 immune response that leads to lesion healing and immunity against the disease. Elucidation of the mechanisms that mediate the strong immunity elicited by L. major will undoubtedly have consequences for vaccine development against all Leishmania species as well as other infections, where strong cell-mediated immune responses are essential for resistance.IL-12 belongs to a family of cytokines including IL-23, IL-27, and ciliary neutrotrophic factor receptor (CNTFR). The bioactive form of this proinflammatory cytokine is a unique heterodimeric protein composed of p35 and p40 subunits that are encoded by...

Human Dendritic Cells Exhibit a Pronounced Type I IFN Signature following Leishmania major Infection That Is Required for IL-12 Induction

“…It has been well established that IL-12 production elicited by human DCs is parasite species-specific, where generally L. major induces IL-12 and L. donovani does not (5, 10). The IL-12p40 promoter contains an Ets (TTTCCT) and a NFκB (GAAATTCCCC) consensus sequence that, upon transcription factor activation, mediates the gene transcription of IL-12p40 (25).…”

“…Total RNA from uninfected or Leishmania- infected DCs was isolated using an RNEasy kit (Qiagen, Valencia, CA) and 1μg of RNA per infection sample was used to generate cDNA using SuperScript III Synthesis (Invitrogen, Carlsbad, CA). For each gene, relative numbers of mRNA copies were determined by the ΔΔC T method (10). Briefly, experimental C T values were normalized to HPRT C T values and depicted as fold change over uninfected samples (calibrator).…”

“…Upon reassessment of the microarray data set, a follow-up kinetic analysis demonstrated that IRF1 and IRF8 genes were also differentially expressed in L. major versus L. donovani infected human DCs. These results indicated that L. major activates IRF1 and IRF8, which subsequently bind to the IL-12p40 and IL-12p35 promoter regions, spurring transcriptional activation through protein complex formation (10). Here, we take a global approach employing microarray technologies to elucidate the modulated host DC transcriptional mechanisms that may influence the robust IL-12 induction observed following L. major infection.…”

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“…In addition, activation of the transcription of NF-kb seems to be crucial in protecting against apoptosis of cells infected with intracellular pathogens [64], [70]- [72]. On the other hand, NF-kb induced IRF-1 activation (figure :9) [73]. This transcription factor is an immunomodulatory factor, it works downstream of the pathogen recognition receptor signaling [74].…”

Interferon Regulatory Factor 7 Contributes to the Control ofLeishmania donovaniin the Mouse Liver

“…Irf-1 is involved in the downregulation of interleukin-12 (IL-12) in dendritic cells (DC) observed following infection with L. amazonensis (41). L. major induces IRF1 and IRF8 in human DC, and their protein prod-ucts bound to the IL-12p35 promoter (15). L. donovani promastigotes stimulate IRF-1 expression in an lipophosphoglycan (LPG)-dependent manner in murine J774.1 cells (2).…”