Allergenic Lipocalins shape dendritic cells to induce TH2 immune responses

Why and when the immune system skews to Th2 mediated allergic immune responses is still poorly characterized. Most of the major as well as minor mammalian allergens causing respiratory sensitization and at least one major food allergen belong to the lipocalin family. With two pairs of homologous recombinant proteins of the lipocalin family, each consisting of an allergen and a non- allergenic human endogenous lipocalin, we investigated their impact on human monocyte derived dendritic cells. Parameters involved in directing the type of immune responses including antigen uptake, maturation induction, tryptophan breakdown and cytokine production by human monocyte derived dendritic cells were measured and the type of immune response induced was characterized in dendritic cell-T cell co-cultures by key cytokine secretion of T cells. Both allergenic lipocalins, Can f 1 (the major respiratory dog allergen) and Fel d 4 (a cat dander respiratory allergen) persistently induced less of the Th1 skewing maturation marker expression, tryptophan breakdown and IL-12 production in human monocyte derived dendritic cells when compared to the endogenous non- allergenic lipocalins Lcn-1 (the human tear lipocalin) and MUP (major urinary protein, produced by the human salivary gland). As a consequence, T cells stimulated by dendritic cells presenting Can f 1 or Fel d 4 produced more of the Th2 signature cytokine IL-13 and less of the Th1 signature cytokine IFN- than T cells stimulated by Lcn-1 or MUP treated dendritic cells. Thus, the crosstalk of dendritic cells with lipocalins has the potential to direct the type of the immune response induced. A global gene expression reactome analysis to investigate the differential effects of the lipocalins on dendritic cells strongly indicates differences in intracellular trafficking, sorting and antigen presentation. Therefore, we propose to investigate in detail the uptake and intracellular transport of allergenic and non- allergenic lipocalins in dendritic cells in aim 1 of this proposal. Of the molecules forming the above cited reactomes we chose formyl peptide receptors (FPR) as a target of aim 2 of this proposal because FPR3 is known to inhibit dendritic cell maturation. Our preliminary data show that FPR3 is strongly expressed in immature dendritic cells and down-regulated by non-allergenic but not by allergenic lipocalins. In aim 3 we propose to direct allergenic lipocalins to the DEC-205 endocytic receptor by fusing the allergens to an anti-DEC-205 antibody because this has been shown to induce Th1 responses or tolerance with several antigens. Investigating the uptake and processing of lipocalins, their interaction with FPR3 and their redirection to the DEC-205 endocytic receptor may, thus, yield new insights into the development of allergic immune responses and possibly novel clues for therapy.

Lipocalins constitute a protein family consisting of small endogenous human proteins and most of the respiratory allergens derived from furry animals. During this project, we first developed a test system allowing to evaluate the allergenic capacity of lipocalins: dendritic cells activated by allergenic or not allergenic lipocalins are co-cultured with nave T helper cells. According to their previous activation, dendritic cells can stimulate the T cells to differentiate into TH2 cells, the major effector cells of allergenic immune responses, or TH1 cells, the hallmark of anti-microbial immunity. To find differences between dendritic cells activated by allergenic and not allergenic lipocalins we assessed their expression profile and found the prominently differentially expressed FPR family. After showing that dendritic cells of this protein family express only FPR3 we investigated the role of this receptor in dendritic cells. We show that FPR3 in dendritic cells interact only with allergenic members of the lipocalin family. This interaction leads to the downregulation of Interleukin 12 expression. Reduced Interleukin 12 is a fundamental requirement for the differentiation of TH2 cells from nave T cells. Inhibiting the function of FPR3 either by interfering which its expression or by the addition of an antagonistic peptide reverts the downregulation of interleukin 12 and consequently, the induction of TH2 cells. Interfering with the function of FPR3 may thus lead to novel therapies for allergies to lipocalins.