Jongdae Lee

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor α and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-κB. However, TLR8 activation by R-848 and TLR2 activation by {S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)} were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

Immunomodulatory functions of type I interferons

Interferon-α (IFNα) and IFNβ, collectively known as type I IFNs, are the major effector cytokines of the host immune response against viral infections. However, the production of type I IFNs is also induced in response to bacterial ligands of innate immune receptors and/or bacterial infections, indicating a broader physiological role for these cytokines in host defence and homeostasis than was originally assumed. The main focus of this Review is the underappreciated immunomodulatory functions of type I IFNs in health and disease. We discuss their function in the regulation of innate and adaptive immune responses, the response to bacterial ligands, inflammasome activation, intestinal homeostasis and inflammatory and autoimmune diseases.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-κB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IκBα degradation and activation of the NF-κB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IκB accumulates in the cytoplasm preventing NF-κB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-κB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

Toll-like receptor 9–induced type I IFN protects mice from experimental colitis

Experimental colitis is mediated by inflammatory or dysregulated immune responses to microbial factors of the gastrointestinal tract. In this study we observed that administration of Toll-like receptor 9 (TLR9) agonists suppressed the severity of experimental colitis in RAG1-/- but not in SCID mice. This differential responsiveness between phenotypically similar but genetically distinct animals was related to a partial blockade in TLR9 signaling and defective production of type I IFN (i.e., IFN-alpha/beta) in SCID mice upon TLR9 stimulation. The addition of neutralization antibodies against type I IFN abolished the antiinflammatory effects induced by TLR9 agonists, whereas the administration of recombinant IFN-beta mimicked the antiinflammatory effects induced by TLR9 agonists in this model. Furthermore, mice deficient in the IFN-alpha/beta receptor exhibited more severe colitis than wild-type mice did upon induction of experimental colitis. These results indicate that TLR9-triggered type I IFN has antiinflammatory functions in colitis. They also underscore the important protective role of type I IFN in intestinal homeostasis and suggest that strategies to modulate innate immunity may be of therapeutic value for the treatment of intestinal inflammatory conditions.

ERK activation drives intestinal tumorigenesis in Apc min/+ mice

Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apcmin/+ mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal–related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apcmin/+/Myd88−/− mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apcmin/+ mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apcmin/+/Myd88−/− mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apcmin/+/Myd88−/− and Apcmin/+ mice and increased their survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host.

3-Hydroxyanthranilic acid inhibits PDK1 activation and suppresses experimental asthma by inducing T cell apoptosis

3-Hydroxyanthranilic acid (HAA), a compound generated during tryptophan metabolism initiated by indoleamine 2,3-dioxygenase, is known to induce T cell death, but its molecular target is not known. Here we report that HAA inhibits NF-κB activation upon T cell antigen receptor engagement by specifically targeting PDK1. Inhibition of NF-κB by HAA leads to dysfunction and cell death of activated Th2 cells, which in turn suppresses experimental asthma. Inhibition of NF-κB and induction of apoptosis is specific to CD4 T cells because HAA does not inhibit NF-κB activation or induce cell death upon Toll-like receptor 4 stimulation in dendritic cells. Thus, HAA is a natural inhibitor that restrains T cell expansion and activation.

TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4+ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10-/-Tlr4-/- mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10-/- and Il10-/-Tlr9-/- mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10-/-Tlr4-/- CD4+ T cells into Rag1-/- recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10-/- CD4+ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4+ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4+ T cell responses.

Conventional dendritic cells regulate the outcome of colonic inflammation independently of T cells.

We explored the physiological role of conventional dendritic cells (cDCs) in acute colitis induced by a single cycle of dextran sodium sulfate administration. Depending on their mode of activation and independently of T cells, cDCs can enhance or attenuate the severity of dextran sodium sulfate-induced colitis. The latter beneficial effect was achieved, in part, by IFN-1 induced by Toll-like receptor 9-activated cDCs. IFN-1 inhibits colonic inflammation by regulating neutrophil and monocyte trafficking to the inflamed colon and restraining the inflammatory products of tissue macrophages. These data highlight a novel role of cDCs in the regulation of other innate immune cells and position them as major players in acute colonic inflammation.

Homeostatic effects of TLR9 signaling in experimental colitis

Abstract:  The commensal microflora of the intestinal tract confer multiple health benefits to the host, including amelioration of inflammatory bowel disease (IBD). Yet, the exact mechanisms by which it ameliorates experimental colitis in animals and human IBD are largely unknown. We tested whether the attenuation of experimental colitis by probiotic bacteria is mediated by toll‐like receptor (TLR) signaling. The severity of colitis was attenuated by delivery of nonviable, γ‐irradiated, or by viable probiotics, but not by heat‐killed probiotics, in wild‐type mice in mice deficient in TLR2 or TLR4. In contrast we did not observe any inhibition of experimental colitis by probiotics, in mice deficient in MyD88 or TLR9. Furthermore, administration of probiotic DNA ameliorated the severity of experimental colitis, whereas methylated probiotic DNA, calf thymus DNA, and Dnase‐treated probiotics had no effect. In subsequent studies, we identified that TLR9‐induced type 1 IFN mediates the anti‐inflammatory effects in experimental colitis. The addition of neutralization antibodies to type 1 IFN abolished the anti‐inflammatory effects, whereas the administration of recombinant IFN‐β mimicked the anti‐inflammatory effects induced by TLR9 agonists.Taken together, these results indicate that the protective effects of probiotics are mainly mediated by their own DNA rather than by their metabolites or their ability to colonize the colon. These findings underscore the diverse effects of indigenous microbial TLR ligands in intestinal homeostasis and intestinal inflammation and suggest that strategies, that modulate type 1 IFN may be of therapeutic value for intestinal inflammatory conditions.

Toll-like receptor signaling in intestinal epithelial cells contributes to colonic homoeostasis.

Purpose of review Since intestinal epithelium expresses Toll-like receptors, it was suggested that the intestinal epithelium is actively involved in the maintenance of colonic homeostasis. Here we describe our recent findings, which support an active contribution of colonic epithelium to intestinal homeostasis via a unique activation of epithelial TLR9. Recent findings Recent data indicate that stimulation of Toll-like receptors by intestinal microbiota supports colonic homeostasis. Several Toll-like receptors are expressed in intestinal epithelium. TLR9, an intracellular protein in immune cells, is expressed on the cell surfaces of intestinal epithelium, both on the apical and the basolateral membrane. TLR9 signaling varies in a domain-specific manner; whereas JNK is activated by TLR9 ligand both apically or basolaterally, NF-κB is activated only via basolateral stimulation. In apical TLR9 stimulation, IκB is phosphorylated and ubiquitinated but is not degraded, and NF-κB-dependent inflammatory signals are not transduced. Stimulation of apical TLR9 compromises the inflammatory cascade induced basolaterally by several other Toll-like receptor ligands, suggesting that apical exposure to luminal microbial DNA restrains intestinal inflammation. Summary These data indicate that certain luminal bacterial products support colonic homeostasis via activation of epithelial Toll-like receptors. The role of epithelial Toll-like receptor expression and activation in the pathogenesis of human inflammatory bowel disease is yet to be explored.