Role of Antimicrobial Peptides in Plasmacytoid Dendritic Cell-driven Inflammation of the Skin

Michel Gilliet, M.D.

M.D. Anderson Cancer Center

Funded in September, 2007: $500000 for 3 years
LAY SUMMARY . ABSTRACT .

LAY SUMMARY

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Identifying the Potential Trigger of Psoriasis, a Chronic Autoimmune Inflammatory Skin Disease

Researchers will determine whether a specific antimicrobial chemical, which is secreted when skin is damaged, triggers immune events that lead to development of psoriasis, an autoimmune inflammatory skin disease. 

Damaged skin cells ordinarily secrete small amounts of a peptide called LL-37, which provides a short-lived attack against invading microbes. When the damaged skin cells die, they also release their DNA. In people who develop psoriasis, however, the levels of LL-37 peptide are higher.  The researchers suspect that the peptide is not effectively regulated, and its persistent anti-microbial activity triggers a series of immune reactions that produce chronic autoimmune skin disease.  Specifically, they hypothesize from extensive preliminary data that is soon to be published in the journal Nature, the LL-37 peptide binds to the DNA released by the patients’ dying skin cells. The patients’ innate immune dendritic cells recognize this as “foreign” DNA, and stimulate adaptive immune T cells to release an inflammatory substance (called “interferon”) against the patients’ own skin cells. This produces chronic skin lesions. 

The researchers will test the hypothesis by undertaking analyses of skin tissue samples from patients and healthy volunteers at both M.D. Anderson and Henrich-Heine University. Additionally, the M.D. Anderson investigators will study immune responses in laboratory animals in which skin from patients or healthy participants have been transplanted. The collaborators will determine whether the LL-37 immune activation pathway is present during common injuries to the skin; whether regulation of LL-37 levels differs in patients compared to volunteers; and how initial immune activation is linked to subsequent induction of inflammation by adaptive immune T cells.

Significance:  The research may lead to the development of effective therapies for psoriasis and potentially other chronic inflammatory diseases.

ABSTRACT

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Role of Antimicrobial Peptides in Plasmacytoid Dendritic Cell-driven Inflammation of the Skin

BACKGROUND
Plasmacytoid dendritic cell (pDC) activation to produce type I IFNs represents a central aspect of immunity in both health and disease. pDCs detect viral genomes via endosomal Toll-like receptor (TLR)-7 and TLR-9 and their production of type I IFNs is critical in anti-viral host defense. PDC-derived type I IFNs are also implicated in the aberrant activation of the adaptive immune system in autoimmunity. We have previously shown that, in psoriasis, a prototype autoimmune disease of the skin, type I IFN production by pDCs triggers T cell-mediated autoimmune skin inflammation leading to disease formation. Yet the factors leading to pDC activation and the pathways involved in their recognition have been unknown.

PRELIMINARY STUDIES
We recently identified LL-37, an endogenous antimicrobial peptide also named human cathelicidin, as the key mediator of pDC activation in psoriatic skin. LL-37 induces robust type I IFN production by converting otherwise non-stimulatory self-DNA into a potent trigger of pDC activation by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Our data suggest a fundamental principle in immune activation by LL-37 through the break of innate tolerance to self-DNA.

HYPOTHESIS/SPECIFIC AIMS
Antimicrobial peptide LL-37 is rapidly and transiently induced by injury to the skin as a result of secretion from keratinocytes and deposition from degranulation of recruited neutrophils. Our data suggest a unique link between skin damage and the activation of the innate immune system through the break of tolerance to self-DNA. In Specific Aim 1 we will therefore determine whether LL-37/self-DNA-driven pDC activation is involved in the transient immune activation during common skin injury.

Psoriasis is typically triggered by skin injury (the Koebner reaction). While LL-37 expression in wounded skin is transient and ceases upon healing, LL-37 appears to be sustained at high levels in psoriatic skin. In Specific Aim 2 we will therefore determine whether injury to psoriatic skin leads to a dysregulated persistent expression of LL-37 and evaluate the mechanisms involved.

pDC activated by LL-37/self-DNA do not differentiate into dendritic cells capable of stimulating T cells. Thus we hypothesize that the LL-37/DNA activation of pDC is linked to productive, adaptive T cell immune responses through the IFN-induced maturation of bystander myeloid dendritic cells (mDC). In Specific Aim 3 we will test this hypothesis.

We have previously shown that pDC activation in psoriatic skin initiates the T cell-mediated autoimmune skin inflammation leading to the formation of lesions. Because LL-37 represents the principal mediator of pDC activation in psoriatic skin, our data suggest a pathogenic link between LL-37 and the development of psoriasis lesions. In Specific Aim 4 we will determine whether the LL-37/self-DNA pathway drives T cell-mediated inflammation in psoriatic skin leading to disease development and assess the role of LL-37 as a novel therapeutic target.

EXPERIMENTAL SETUP
To explore Specific Aim 1, skin of healthy individuals will be physically or chemically irritated by tape stripping or application of contact irritant SLS, respectively to induce a mild skin injury. Skin biopsies will be obtained from individual subjects at 0, 6, 24 and 48 hours following skin injury, so that the time courses of cellular infiltration and cytokine expression can be charted. We will track the numbers of pDCs, myeloid DC and T cells and correlate the data with the expression of LL-37. We will also measure the expression of many cytokines including type I IFNs. This will be accomplished by using techniques including double, sequential immunohistochemistry (IHC) and real-time PCR.

To explore Specific Aim 2, we will take advantage of an established xenograft model of human psoriasis in which uninvolved skin from a psoriasis patient converts into psoriatic skin upon transplantation onto AGR mice. We will determine whether LL-37 induced in engrafted skin by the transplantation procedure is sustained during the development of psoriasis and compare it to the expression of LL-37 in engrafted healthy skin.

To explore Specific Aim 3, we will establish a pDC-mDC coculture system stimulated by LL-37/self-DNA complexes. We will evaluate DC-survival, maturation, cytokine expression, as well as ability to stimulate and differentiate T cells in-vitro.

To explore Specific Aim 4 we will block LL-37 activity in the xenograft model of human psoriasis by using specific neutralizing antibodies. We will assess whether we can inhibit pDC-derived IFN-a in engrafted skin and consequently block the initiation of T cell mediated skin inflammation and the development of psoriatic lesions.

Significance: The data obtained from these experiments will allow us to establish a role of LL-37 in inflammation in wounded skin and a pathogenic role of LL-37 in psoriatic autoimmune inflammation. This will open new avenues for therapeutic intervention in psoriasis and potentially other chronic inflammatory diseases.