Blake C. Jones, Benjamin C. Chaon, Jennifer M. Enright, Daniel M. Koehler, Lisa A. Zickuhr, and Mary Ann McDowell Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556

Fetal Skin Dendritic Cells Do Not Mimic Human Dendritic Cells in Response to Leishmania major Infection

Western Blot

Figure 6. Nuclear extracts from FSDC experimental groups and from L. major V1. Western blot was performed using p50, p65, and c-Rel antibodies. Lamin A/C and Hsp90 served as nuclear and cytosolic markers, respectively. Nuclear translocation of p50 was observed only after 60 minutes. Cleavage of p65 and c-Rel was observed in the V1 + LPS and V1 + LPS + IFN-γ conditions.

Berthier-Vergnes O, Bernard F, Vincent F, Massacrier C, Schmitt D, Pequet-Navarro J. 2005. TNF-α ehances phenotypic and functional maturation of human epidermal Langerhans cells and induces IL-12 p40 and IP-10/CXCL-10

production. FEBS Letters 579:3660-3668.

Cameron P, McGachy A, Anderson M, Paul A, Coombs G, Mottram J, Alexander J, Plevin R. 2004. Inhibition of lipopolysaccharide-induced macrophage IL-12 production by L. mexicana amastigotes: the role of cysteine peptidases in the NF-kappa B signaling pathway. Journal of Immunology 173:3297-3304.

Guizani-Tabbane L, Ben-Aissa K, Belghith M, Sassi A, Dellagi K. 2004. Leishmania major amastigotes induce p50/c-Rel NF-κB transcription factor in human macrophages: involvement in cytokine synthesis. Infection and Immunity 2582-2589.

Murphy TL, Cleveland MG, Kulesza P, Magram J, Murphy KM. 1995. Regulation of interleukin-12 p40 expression through an NF-kappa B half-site. Mol Cell Biol. (10):5258-67.

Tato CM, Hunter CA. 2002. Host-pathogen interactions: subversion and utilization of the NF-kappa B pathway during infection. Infect. Immun. 70(7):3311-7.

Wolf R.A. Retrieved fromhttp://www.bio.davidson.edu/COURSES/Immunology/Students/spring2000/wolf/tnfalpha.html. 29 Apr 2006.

Works Cited

Acknowledgments

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania spp. Leishmania major is a member of this genus that causes the formation of self-healing cutaneous lesions. L. major is an obligate intracellular parasite and is transferred to its host during the blood meal of a female sand fly. The parasites live and reproduce within the phagosomes of macrophage and dendritic cells as amastigotes.

The necessary immune response for parasite elimination is the TH1 response, which is characterized by the secretion of cytokines such as IL-12, IFN-γ and TNF-α. Bioactive IL-12 is a 70kDa heterodimer composed of p35 and p40 subunits. Among its many functions, it has the ability to stimulate CD4+ cells to differentiate into TH1 cells. TH1 cells produce IFN-γ that, in turn, functions in a positive-feedback mechanism to further induce IL-12 transcription and the TH1 response.

IL-12

Production of IL-12 is directed by the transcription factor NF-κB. NF-κB is bound to IκB in the cytoplasm, and, upon stimulation, it is released and translocated to the nucleus. Previous research suggests the p50, c-Rel, and p65 monomers play an important role in IL-12 transcription upon L. major infection. Bacterial lipopolysaccharide (LPS) has also been found to induce NF-κB translocation and IL-12 transcription and secretion. TNF-α is also regulated by NF-κB.

Introduction

Conclusions

Experimental Design

Future Experiments

AbstractLeishmania species are intracellular parasites infecting host macrophages resulting in a broad range of devastating diseases in 88 countries. Leishmania major is the causative agent of cutaneuos leishmaniasis which results in an ulcerative lesion forming on the skin; this lesion is usually self healing. Dendritic cells play a vital role in pathogen recognition and initiation of the appropriate immune response. Dendritic cells secrete pro-inflammatory cytokines such as IL-12 and TNF-α which are necessary for successful clearance and immunity to L. major. Previous research using human dendritic cells infected with L. major has shown these cells to produce large amounts of IL-12. Fetal skin dendritic cells (FSDC) are a cell line derived from fetal mouse skin; these cells are known to be dendritic cell precursors and should respond to L. major infection similar to human dendritic cells. Interestingly, when we assessed IL-12 and TNF-α production in FSDC upon infection with L. major we found that FSDC do not produce IL-12 however large amounts of TNF-α are produced.

We would like to acknowledge the assistance of: Rachel Kasuboski, Dr. Mary Ann McDowell, McDowell Lab (Michael Donovan, Cristina Carter, Asha Jayakumar, James Whitcomb), Dr. Michelle Whaley and the BIOS 27241 teaching staff, Marri Kajfez, David Choate, and the Ferdig, Adams, Collins, and O’Tousa Labs.

Metacyclic Promastigotes

Infection into FSDC

Experimental Groups

Flow cytometry detection of bioactive IL-12p70, p40, and TNF-α in all experimental groups to determine the effects of L. major infection on cytokine storage in FSDC murine dendritic cell precursors.

Immunofluorescent detection of IL-12 and TNF-α receptor on FSDC cells to determine if an autocrine IL-12 or TNF-α response is occurring.

Differentiate FSDC cells with CD40 ligand into mature dendritic cells to determine if these dendritic precursor cells are too immature to produce and secrete IL-12.

EMSA to determine if NF-κB is binding to IL-12 promoter regions of IL-12 .

Investigate the effects of L. major infection on TNF-α transcription through quantitative real-time PCR.

Repeat experiments using primary murine dendritic cells.

M

L. m

ajor

p50 (60min)

Hsp 90 (40min)

Lamin A/C (40min)

c-Rel (40min)

p65 (40min)

p50 (40min)

50 kDa

65 kDa

35 kDa75 kDa

35 kDa69 kDa

90 kDa

50 kDa

LPS LPS + IFN-γ

M LPS LPS + IFN- γ

M LPS LPS + IFN- γ

Uninfected Infected Heat-Killed

ELISAto determine if L. major infection

affects secretion of IL-12 p40 and p70 and TNF-α

Real-Time PCRto determine if L. major infection affects transcription levels of IL-

12 p40 and p70

Western Blotto determine if L. major infection inhibits nuclear translocation or

causes degradation of NF-κB

Flow Cytometry

to determine if IL-12 and TNF-α are contained within the FSDC cells

Culture FSDC Cells and L. major ParasitesInfect and Stimulate Experimental Groups

Western blotting revealed that infection induces a small amount of NF-κBp65 and c-Rel nuclear translocation upon infection in unstimulated cells.

LPS and LPS + IFN-γ stimulation increase NF-κBp65 and c-Rel nuclear translocation in both infected and heat-killed groups.

Cleavage of NF-κBp65 and c-Rel was observed in infected experimental groups stimulated with LPS and LPS + IFN-γ, similar to what is observed in human dendritic cells.

RT-PCR showed that LPS and LPS + IFN-γ are significant inducers of IL-12 p40 transcription.

Upon infection, L. major causes a reduction in IL-12 transcription.

Flow cytometry demonstrated intracellular storage of the TH1-associated cytokines IL-12p70 and TNF-α.

ELISA indicates that FSDC cells do not secrete IL-12p70 but do secrete biologically significant levels of TNF-α upon stimulation.

FSDC cells do not mimic human dendritic cells with regard to an IL-12 response.

L. major

IL-12

X

TH1 ResponseTH1

Dendritic Cell

IFN-γ

NF-κB Nuclear Translocation

ELISA Figure 2. p70, p40, and TNF-α secretion by FSDC cells infected with L. major strain V1. No biologically significant secretion of IL-12p70 or p40 was observed. A significant decrease in TNF-α secretion was observed between infected and uninfected conditions that were stimulated with LPS only as well as LPS + IFN-γ. *p<0.05

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p40 Secretion (48h) p70 Secretion (48h)

Infection Rates

Figure 1. Infection rates for all experiments. Infection rates were determined by counting an average of 200 cells (uninfected + infected) and the intracellular and surface parasites. These results indicated successful infections.

Media LPS LPS + IFN- Media LPS LPS + IFN-0

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Western Blot

Cell culture: The FSDC murine dendritic cell line was the generous gift of Paola Ricciardi-Castagnoli. Cells were cultured in Iscove’s DMEM media (5% heat inactivated FBS, 100 IU/mL penicillin, 100 mg/mL streptomycin, 2mM L-glutamine, 50μM β-mercaptoethanol) at 5% CO2, and 37° C.

Parasite culture and infection protocol: Leishmania major parasites (strain V1) were cultured in M199 complete medium (10% heat inactivated FBS, 100μg/mL penicillin, 100μg/mL streptomycin, 2mM L-glutamine, 40mM HEPES, 0.1mM adenine (in 50mM HEPES), 5mg/mL hemin (in 50% triethanolamine) and 1 mg/mL biotin) at 25°C. 5-6 day stationary phase metacyclic promastigotes (infective stage) were isolated for infection into FSDC cells using a ficoll density gradient. Infections were performed by plating FSDC cells and infecting with a 10:1 promastigote to host cell ratio after two hours. IFN-γ groups received IFN-γ (1:2000) 2 hours post infection, LPS groups were stimulated with LPS (1:2000) at 4 hours post infection, and LPS + IFN-γ groups received both. A small fraction of the infected cells were prepared and fixed to slides via cytocentrifugation and differentially stained with DiffQuick. Infection rate was determined by counting an average of 200 cells and both intracellular and attached surface parasites to generate parasite number per 100 cells.

ELISA: Supernatants were collected at 24 and 48 hours post-stimulation. ELISA for the p40 monomer and bioactive p70 dimer of Interleukin-12 was performed using monoclonal anti-mouse IL-12 p40 and p70 coating antibodies (Endogen) with biotin labeled p40/p70 detecting antibodies (Endogen). TNF-α ELISA was performed using the BD OptEIATM mouse TNF ELISA Set (BD Biosciences). A Student’s T-test was used for statistical comparison and a p<0.05 was considered significant. Western Blot: Nuclear extracts were isolated from experimental groups using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology). An equivalent of 8x105 cells (40μl) was separated by SDS-PAGE and transferred to a PVDF membrane using the Novex system (Invitrogen Life Technologies). Ponceau and Coomassie (BD Biosciences) staining were performed to ensure successful protein transfer. Blots were probed with monoclonal mouse anti-NF-κBp50, anti-NF-κBp65, and anti-NF-κB-cREL 1° antibodies (Santa Cruz Biotechnology). The nuclear and cytoplasmic markers, LaminA/C and HSP90 respectively, were detected using monoclonal mouse-anti-HSP90 and polyclonal goat-anti-LaminA/C antibodies. Secondary polyclonal HRP-conjugated goat-anti-mouse and donkey anti-goat antibodies (BD Biosciences) were used in conjunction with the SuperSignal West Femto Maximum Sensitivity Substrate and SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).

RNA Isolation and Generation of cDNA: RNA was extracted from experimental groups using the RNeasy RNA Isolation Kit (Qiagen). DNA was removed from RNA by treatment with DNAse I (Invitrogen). This purified RNA was used as the template in a subsequent reverse-transcription reaction using SuperScript III Kit (Invitrogen) with random primers and dNTPs (Invitrogen) to generate cDNA.

Quantitiative Real-Time PCR: Quantitative real-time PCR was performed on the cDNA using the 2x SYBR-Green Kit (Applied Biosystems) to amplify the p40 monomer of Interkeukin-12 and the housekeeping gene HPRT. Reactions were run on the ABI 7900HT Sequence Detection System. Primers used, IL-12 p40, 5’-AAC CAT CTC CTG GTT TGC CA-3’ and 5’-CGG GAG TCC AGT CCA CCT C-3’ and HPRT, 5’-CAA AGC CTA AGA TGA GCG CAA-3’ and 5’-AGG CAG ATG GCC ACA GGA C-3’ (Integrated DNA Technologies). Relative copy number was determined by: # of copies = 2-ΔΔCt, ΔΔCt = Δct (experimental) – Δct (calibrator), Δct = Δct (experimental) – Δct (HPRT), ct = cycle at which a statistically significant increase in the emission intensity over background was detected, and Δct (calibrator) = the mean Δct for the naïve control. A Student’s T-test was used for statistical comparison with p<0.05 considered significant. Flow Cytometry: FSDC cells were plated and stimulated overnight with IFN-γ. Subsequently the cells were stimulated with LPS for one hour and then treated with Brefeldin-A for six hours to block secretion. The cells were blocked and permeablized overnight with BD Perm/Wash Buffer (BD Biosciences) with 10% Normal Mouse Serum. Cells were stained with PE-conjugated rat-anti-mouse monoclonal TFN-α and bioactive IL-12 p40/p70 antibodies (BD PharMingen) and analyzed on the Cytomics FC 500 MPL (Beckman Coulter).

Materials and Methods Flow Cytometry

Cytokine Production

Figure 3. Percent of cells in which TNF-α and bioactive IL-12p70 cytokines were detected. FSDC cells contain bioactive IL-12 and TNF-α upon stimulation with LPS, IFN-γ, or both.

Media LPS IFN- LPS+IFN-0

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RT-PCR

Figure 4. Expression of IL-12p40 mRNA 8 hours post-infection with L. major V1. p40 transcription levels were significantly increased in LPS and LPS + IFN-γ stimulated conditions compared to media. V1 infection significantly decreased p40 transcription relative to uninfected in the LPS stimulated condition. *p<0.05

IL-12 p40 mRNA

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All lines indicate significance with p<0.05HK + LPS + IFN- γV1 + LPS + IFN- γLPS + IFN-γLPS/IFN-γ costimulation

HK + LPSV1 + LPSLPSLPS stimulation

HKV1MediaUnstimulated

Heat-KilledL. major V1 InfectedUninfected