We have identified a novel secreted protein encoded by an uncharacterized gene (C17Orf99) that is specifically expressed in the fetal liver, bone marrow and activated B cells. The gene sequence predicts a ~27KDa secreted protein with a signal peptide and exhibits no homology to any known cytokine family. However, it is strongly induced upon activation in several B cell lines or in normal B cells activated with stimuli such as CD40L, IL-4 or Lipopolysaccharide (LPS). These characteristics (small secreted protein, expression restricted to activated lymphoid cells) suggest that this gene encodes a novel cytokine which we have named Interleukin-40. In this proposal, we aim to functionally characterize IL-40. We have cloned and expressed mouse and human IL-40, and will use these recombinant proteins to explore its biological functions. We have produced monoclonal antibodies against IL-40 peptides which we are currently screening with recombinant protein. We have also obtained an IL-40-/- mouse that exhibits abnormalities in the Peyer's patches and low levels of IgA in the mammary gland and feces.
In Specific Aim 1, we will undertake the biological characterization of IL-40 in vitro by identifying the cellular sources of IL-40. We hypothesize that IL-40 is produced by certain B cell subsets. We will next determine the cells that produce IL-40 in the fetal liver and bone marrow. These results will help us understand its biological function in these organs. We hypothesize that IL-40 production is linked to the production of IL-4. We will therefore explore whether other strong IL-4 producing cells (like iNKT and Th2 CD4+ cells) also produce IL-40. IL4 is a B cell costimulatory factor that induces proliferation in combination with various B cell mitogens, and these are the same conditions that induce IL-40 production by B cells. We therefore hypothesize that IL-40 may be involved in some of the known biological activities of IL-4. We will test this by performing IL-4 driven proliferation and differentiation assays using either IL-40-/- or wild type (WT) mouse B cells.
In Specific Aim 2, we will explore the physiology of IL-40 using an IL-40-/- mouse, which are viable and fertile. We will explore the Peyer's patches of these mice to explore the cellular changes that have taken place due to the lack of IL-40, and we will quantify the number of IgA-producing cells in the Peyer's patches of IL-40-/- mice. We will measure the expression of other genes that have been shown to influence IgA expression like CCL28 and its receptor CCR10, as well as TGF?. IgA levels are linked to the gut microbiome, so we will analyze the gut microbiome of the IL-40-/- mouse for abnormalities. Finally, we will search for molecular defects that may affect the production of Immunoglobulin production including class switch recombination in the B cells of the IL-40-/- mouse. Through these specific aims we will open a new field of research, namely, the study of the biology of IL-40.
We have discovered a novel cytokine (Interleukin-40: IL-40) and we hypothesize that it is a novel immunoregulatory molecule. Using recombinant IL-40 and an IL-40 deficient (knockout) mouse we will undertake the functional characterization of IL-40 in vitro and in vivo.
|Catalan-Dibene, Jovani; Vazquez, Monica I; Luu, Van Phi et al. (2017) Identification of IL-40, a Novel B Cell-Associated Cytokine. J Immunol 199:3326-3335|
|Ushach, Irina; Zlotnik, Albert (2016) Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage. J Leukoc Biol 100:481-9|
|Vazquez, Monica I; Catalan-Dibene, Jovani; Zlotnik, Albert (2015) B cells responses and cytokine production are regulated by their immune microenvironment. Cytokine 74:318-26|