Knut Kotarsky

Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids.

Short-chain fatty acids (SCFAs) have long been known to exert cellular effects on blood leukocytes. Acetate, propionate, and butyrate represent the most capable SCFA, inducing calcium mobilization which subsequently regulates leukocyte function in the immune system. We have cloned the previously described putative orphan G-protein coupled receptor, GPR43, and have functionally identified SCFA as the activating ligands. Acetate and propionate were found to be the two most potent ligands, although butyrate, formate, and valerate (in this order of potency) also were able to induce receptor activation. Both the human and mouse receptor homologues were found to share the same pattern of ligand activation. This finding, together with a high degree of amino acid sequence similarity between the mouse and human homologues, indicates an evolutionary conserved function. Upon ligand stimulation, the receptor mobilized intracellular calcium in both a recombinant system as well as in human granulocytes. We found the human gene to be predominantly expressed in peripheral blood leukocytes and, to a lesser extent, in spleen. We suggest the designation FFA(2)R to this second receptor activated by free fatty acids. The first-described FFAR, now named FFA(1)R, is activated by medium- to long-chain free fatty acids.

Lysophosphatidic Acid Binds to and Activates GPR92, a G Protein-Coupled Receptor Highly Expressed in Gastrointestinal Lymphocytes

Here, the ligand binding, activation, and tissue distribution of the orphan G protein-coupled receptor (GPCR) GPR92 were studied. GPR92 binds and is activated by compounds based on the lysophosphatidic acid (LPA) backbone. The binding of LPA to GPR92 was of high affinity (KD = 6.4 ± 0.9 nM) and led to an increase in both phosphoinositide hydrolysis and cAMP production. GPR92 is atypical in that it has a low sequence homology with the classic LPA1-3 receptors (21-22%). Expression of GPR92 is mainly found in heart, placenta, spleen, brain, lung, and gut. Notably, GPR92 is highly expressed in the lymphocyte compartment of the gastrointestinal tract. It is the most abundant GPCR activated by LPA found in the small intestinal intraepithelial CD8+ cytotoxic T cells.

Bile retinoids imprint intestinal CD103+ dendritic cells with the ability to generate gut-tropic T cells

Small intestinal lamina propria (SI-LP) CD103+ dendritic cells (DCs) are imprinted with an ability to metabolize vitamin A (retinol), a property underlying their enhanced capacity to induce the gut-homing receptors CC chemokine receptor-9 and α4β7 on responding T cells. In this study, we demonstrate that imprinting of CD103+ DCs is itself critically dependent on vitamin A and occurs locally within the small intestine (SI). The major vitamin A metabolite retinoic acid (RA) induced retinol-metabolizing activity in DCs both in vitro and in vivo, suggesting a direct role for RA in this process. Consistent with this, SI-LP CD103+ DCs constitutively received RA signals in vivo at significantly higher levels than did colonic CD103+ DCs. Remarkably, SI CD103+ DCs remained imprinted in mice depleted of dietary but not of systemic retinol. We found that bile contained high levels of retinol, induced RA receptor-dependent retinol-metabolizing activity in bone marrow-derived DCs, and imprinted these cells with the ability to generate gut-tropic T cells. Taken together, these results suggest a novel and unexpected role for bile in SI-LP CD103+ DC imprinting.

CCR2(+)CD103(-) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells.

The identification of intestinal macrophages (mφs) and dendritic cells (DCs) is a matter of intense debate. Although CD103+ mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103− MPs remain controversial. We show here that intestinal CD103−CD11b+ MPs can be separated clearly into DCs and mφs based on phenotype, gene profile, and kinetics. CD64−CD103−CD11b+ MPs are classical DCs, being derived from Flt3 ligand-dependent, DC-committed precursors, not Ly6Chi monocytes. Surprisingly, a significant proportion of these CD103−CD11b+ DCs express CCR2 and there is a selective decrease in CD103−CD11b+ DCs in mice lacking this chemokine receptor. CCR2+CD103− DCs are present in both the murine and human intestine, drive interleukin (IL)-17a production by T cells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2+ DCs that is involved in priming mucosal T helper type 17 (Th17) responses.

Differential homing mechanisms regulate regionalized effector CD8αβ+ T cell accumulation within the small intestine

The CC chemokine receptor (CCR)9 is expressed on the majority of small intestinal, but few colonic, T cells, whereas its ligand CCL25 is constitutively expressed by small intestinal epithelial cells. As such, CCR9/CCL25 have been proposed to play a central role in regulating small intestinal but not colonic immune responses and thus to organize regionalized immunity within the intestinal mucosa. Here, we demonstrate that CCL25 is expressed at reduced levels by epithelial cells in the distal compared with proximal small intestine, which correlated with less efficient CCR9-dependent effector CD8αβ+ T cell entry into the ileal epithelium. In vitro-generated α4β7+ effector CD8αβ+ T cell entry into the lamina propria was less dependent on CCR9 than entry into the epithelium along the entire length of the small intestine and in particular in the ileum. CCR9-independent α4β7+ effector CD8αβ+ T cell entry was pertussis toxin-sensitive, suggesting a role for additional GαI-linked G protein-coupled receptors. Finally, in vivo-primed effector CD8αβ+ T cells displayed regionalized differences in their entry to the small intestinal epithelium with enhanced CCR9-independent entry to the ileum. These results highlight a hitherto underappreciated compartmentalization of immune responses within the small intestine and have direct implications for targeting strategies aimed at regulating T cell localization to the small intestinal mucosa.

Functional Characterization of the CCL25 Promoter in Small Intestinal Epithelial Cells Suggests a Regulatory Role for Caudal-Related Homeobox (Cdx) Transcription Factors

The chemokine CCL25 is selectively and constitutively expressed in the small intestinal epithelium and plays an important role in mediating lymphocyte recruitment to this site. In this study, we demonstrate that CCL25 expression in murine small intestinal epithelial cells is independent of signaling through the lymphotoxin β receptor and is not enhanced by inflammatory stimuli, pathways involved in driving the expression of most other chemokines. We define a transcriptional start site in the CCL25 gene and a region −141 to −5 proximal of exon 1 that is required for minimal promoter activity in the small intestinal epithelial cell lines, MODE-K and mICc12. These cell lines expressed far less CCL25 mRNA than freshly isolated small intestinal epithelial cells indicating that they are missing important factors driving CCL25 expression. The CCL25 promoter contained putative binding sites for the intestinal epithelial-associated Caudal-related homeobox (Cdx) transcription factors Cdx-1 and Cdx-2, and small intestinal epithelial cells but not MODE-K and mICc12 cells expressed Cdx-1 and Cdx-2. EMSA analysis demonstrated that Cdx proteins were present in nuclear extracts from freshly isolated small intestinal epithelial cells but not in MODE-K or mICcl2 cells, and bound to putative Cdx sites within the CCL25 promoter. Finally, cotransfection of MODE-K cells with Cdx transcription factors significantly increased CCL25 promoter activity as well as endogenous CCL25 mRNA levels. Together these results demonstrate a unique pattern of regulation for CCL25 and suggest a role for Cdx proteins in regulating CCL25 transcription.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αβ(+) and CD4(+)CD8αα(+) T cells; the latter requiring β8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

A novel role for constitutively expressed epithelial-derived chemokines as antibacterial peptides in the intestinal mucosa.

Intestinal-derived chemokines have a central role in orchestrating immune cell influx into the normal and inflamed intestine. Here, we identify the chemokine CCL6 as one of the most abundant chemokines constitutively expressed by both murine small intestinal and colonic epithelial cells. CCL6 protein localized to crypt epithelial cells, was detected in the gut lumen and reached high concentrations at the mucosal surface. Its expression was further enhanced in the small intestine following in vivo administration of LPS or after stimulation of the small intestinal epithelial cell line, mICc12, with IFNγ, IL-4 or TNFα. Recombinant- and intestinal-derived CCL6 bound to a subset of the intestinal microflora and displayed antibacterial activity. Finally, the human homologs to CCL6, CCL14 and CCL15 were also constitutively expressed at high levels in human intestinal epithelium, were further enhanced in inflammatory bowel disease and displayed similar antibacterial activity. These findings identify a novel role for constitutively expressed, epithelial-derived chemokines as antimicrobial peptides in the intestinal mucosa.

Mesenchymal Cells Regulate Retinoic Acid Receptor-Dependent Cortical Thymic Epithelial Cell Homeostasis.

The vitamin A metabolite and transcriptional modulator retinoic acid (RA) is recognized as an important regulator of epithelial cell homeostasis in several tissues. Despite the known importance of the epithelial compartment of the thymus in T cell development and selection, the potential role of RA in the regulation of thymic cortical and medullary epithelial cell homeostasis has yet to be addressed. In this study, using fetal thymus organ cultures, we demonstrate that endogenous RA signaling promotes thymic epithelial cell (TEC) cell-cycle exit and restricts TEC cellularity preferentially in the cortical TEC compartment. Combined gene expression, biochemical, and functional analyses identified mesenchymal cells as the major source of RA in the embryonic thymus. In reaggregate culture experiments, thymic mesenchyme was required for RA-dependent regulation of TEC expansion, highlighting the importance of mesenchyme-derived RA in modulating TEC turnover. The RA-generating potential of mesenchymal cells was selectively maintained within a discrete Ly51intgp38+ subset of Ly51+ mesenchyme in the adult thymus, suggesting a continual role for mesenchymal cell-derived RA in postnatal TEC homeostasis. These findings identify RA signaling as a novel mechanism by which thymic mesenchyme influences TEC development.

Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.

Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-kappaB, STAT, and AP-1) and hence efficiently funnels several signaling pathways. The assay, performed with the resulting reporter cell line HFF11, has an exceptional high Z-factor and a large signal-to-background ratio. To facilitate cell handling during screening, we introduced a secreted Renilla luciferase as a reporter enzyme. HR36 reporter cells, equipped with the construct, were added to ligands present in a multiwell plate and after addition of coelenterazine they produced a luminescence readout. This procedure economizes cell handling and at the same time increases assay quality and sensitivity