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. 2011 Aug;131(8):1650-9.
doi: 10.1038/jid.2011.99. Epub 2011 Apr 28.

A role for leukocyte-derived IL-1RA in DC homeostasis revealed by increased susceptibility of IL-1RA-deficient mice to cutaneous leishmaniasis

Kordula Kautz-Neu  1 , Susanna Lopez Kostka, Stephanie Dinges, Yoichiro Iwakura, Mark C Udey, Esther von Stebut
Affiliations

A role for leukocyte-derived IL-1RA in DC homeostasis revealed by increased susceptibility of IL-1RA-deficient mice to cutaneous leishmaniasis

Kordula Kautz-Neu et al. J Invest Dermatol. 2011 Aug.

Abstract

Dendritic cell (DC)-derived IL-1α/β plays a critical role in the induction of T helper type 1 (Th1)-dependent immunity against Leishmania. DCs from susceptible BALB/c mice produce less IL-1α/β when compared with resistant C57BL/6 mice, contributing to aberrant Th2 development and ultimate death of infected mice. We have extended our studies of the role of IL-1 in leishmaniasis using IL-1RA(-/-) BALB/c mice that are characterized by upregulated IL-1 receptor signaling. Unexpectedly, infection of IL-1RA(-/-) mice led to significantly worsened disease outcome with larger lesions, dramatically higher parasite burdens, and decreased IFN-γ production by antigen-specific T cells. We determined that IL-1RA(-/-) DCs were more mature already in the steady state, exhibited less phagocytotic capacity, and IL-12 production in response to various stimuli was impaired. Our data suggest that in addition to effects on Th education, IL-1α/β signaling also modulates DC homeostasis with increased signaling, leading to downmodulation of IL-12 synthesis and worsened disease outcome after infection with Leishmania major. Thus, the complex regulation of various members of the IL-1 cytokine family mediated through effects on both DCs and T cells critically contributes to disease outcome against this important human pathogen.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1.
Figure 1.. BALB/c mice deficient in IL-1 receptor antagonist (IL-1RA) exhibit increased susceptibility in cutaneous leishmaniasis because of decreased levels of IL-12 and IFN-γ.
Groups of ⩾5 IL-1RA−/− or BALB/c mice were infected with 103 Leishmania major. (a) Lesion development was assessed weekly. (b) In weeks 6 and 8, lesional and splenic parasite burdens were determined using limiting dilution. Dots represent parasites in individual ears; bars indicate means. (c) Draining lymph nodes were plated at 1 × 106 cells per well and restimulated in the presence of soluble Leishmania antigen (SLA, 25 μg ml−1). All data are expressed as mean±SEM (n⩾8 from seven independent experiments; †mice were killed, *P⩽0.05, **P⩽0.005, and ***P⩽0.002).
Figure 2.
Figure 2.. Th cells from IL-1RA−/− and BALB/c mice are differentially primed in vivo.
(a) Bone marrow-derived dendritic cells (DCs) from BALB/c mice were plated at 2 × 105 per ml and incubated for 18 hours with soluble Leishmania antigen (SLA), Leishmania major amastigotes (Am., five parasites per cell), or Staphylococcus enterotoxin B (SEB, 20 μg ml−1), or left untreated. CD4+ T cells from lymph nodes (LNs) from 6-week-infected IL-1RA−/− and BALB/c mice were enriched and co-cultured with DCs for 48 hours. Supernatants were analyzed for cytokine production by ELISA. Pooled data from two independent experiments are shown (mean±SEM, n = 8 different mice). (b) For intracellular cytokine analysis, LN cells from infected mice were restimulated using phorbol 12-myristate 13-acetate (PMA)/ionomycin in the presence of Brefeldin A for 4–6 hours, followed by extracellular staining for CD3, CD45, and CD4 and intracellular cytokine staining. Representative plots of cells pregated on CD3, CD45, and CD4 are shown (upper panel). Pooled data from two independent experiments are shown (lower panel) (mean±SEM, n = 8 different mice). (a, b) *P⩽0.05, **P⩽0.005, and ***P⩽0.002.
Figure 3.
Figure 3.. IL-1RA−/− dendritic cells (DCs) are more mature and impaired in IL-12 production and in their ability to phagocytose Leishmania major.
Skin-derived macrophages (MΦs) and bone marrow-derived DCs (BMDCs) were plated at 2 × 105 per ml and incubated for 18 hours with lipopolysaccharide (LPS; 100 ng ml−1), IFN-γ (1,000 U ml−1), amastigotes (Am.), or promastigotes (Prom.) of L. major (five parasites per cell) as indicated. (a, b) Cytokine content in supernatants was determined by ELISA. (c) After 18 hours, cells were harvested and infection rates as well as the number of parasites/cell were determined on DiffQuick-stained cytospins. All data are expressed as mean±SEM (n⩾4; *P⩽0.05). (d, e) DCs were generated from bone marrow using rGM-CSF and IL-4 (10 ng ml−1). In some cultures, rhIL-1RA was added at 1 μg ml−1. On day 6, cells were harvested and analyzed for expression of major histocompatibility complex (MHC) class II, CD40, CD54, and CD86 by flow cytometry. (d) One representative staining with mean fluorescence intensity (MFI) is shown. (e) Pooled data from n⩾4 independent experiments are shown. IL-1RA−/− MFI values were normalized to 100% and relative changes in other groups calculated (mean±SEM; *P⩽0.05, and ***P⩽0.002).
Figure 4.
Figure 4.. Restoration of a normal immature phenotype of IL-1RA−/− BALB/c dendritic cells (DCs) with recombinant IL-1RA.
(a, b) Bone marrow-derived DCs (BMDCs) were generated with rGM-CSF and IL-4 (10 ng ml−1 each) and harvested as immature DCs on day 6. (c, d) CD11c+ DCs were isolated ex vivo from lymph nodes (LNs) and spleens using MACS beads. All cells were plated at 2 × 105 cells per ml and stimulated with lipopolysaccharide (LPS; 100 ng ml−1), IFN-γ (1,000 U ml−1), and Leishmania major amastigotes (Am., 1:5 cells/parasites). (a, b) In addition, some co-cultures were treated with rhIL-1RA during culture (IL-1RAsubst. cult., 1 μg ml−1) and/or restimulation (IL-1RArestim, 50 ng ml−1). (a, d) IL-12p40 release was determined by ELISA. (b) Infection rates of DCs were assessed on cytospins. (c) Spleen DCs were analyzed for their expression of major histocompatibility complex (MHC) class II using flow cytometry. One representative of two independent experiments is shown, numbers in the upper right corner represent mean fluorescence intensity (MFI) values. (a, b, d), Pooled data from n⩾5 independent experiments are shown (mean±SEM; *P⩽0.05, and **P⩽0.005).
Figure 5.
Figure 5.. Leishmania susceptibility of IL-1RA−/− BALB/c mice is determined by bone marrow (BM)-derived cells.
IL-1 receptor antagonist (IL-1RA)-deficient or wild-type mice were lethally irradiated with 7 Gy, adoptively transferred with either wild-type or IL-1RA−/− BM (5 × 106 cells per mouse intravenously (i.v.)), and rested for 6 weeks. Subsequently, mice were infected with physiologically relevant low-dose inocula of Leishmania major (103 metacyclic promastigotes). (a) Lesion development was assessed in three dimensions. In week 6, lesional parasite burdens were determined using a limiting dilution assay. Dots represent parasites in individual ears, bars indicate means. (b) In week 6, draining lymph nodes were harvested and restimulated with soluble Leishmania antigen (SLA; 1 × 106 per ml). Cytokine release into 48 hours of supernatants was determined by ELISA. Pooled data from n = 2 independent experiments are shown (mean±SEM; n⩾8, *P⩽0.05, **P⩽0.005, and ***P⩽0.002). * Differences between BALB/c recipient mice; #differences between IL-1RA−/− recipients.
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