Hepatocytes induce Foxp3⁺ regulatory T cells by Notch signaling.

The liver plays a pivotal role in maintaining immunological tolerance, although the exact molecular mechanism is still largely unknown. The induction of systemic tolerance by liver resident APCs has been attributed to peripheral deletion and to the induction of Tregs. HCs, the parenchymal cells in the liver, could function as nonprofessional APCs and interact and establish cell-cell contact with T lymphocytes. We hypothesized that HCs from healthy or regenerated livers may contribute to induction of functional Tregs. Here, we show that murine HCs induced Foxp3(+) Tregs within CD4(+) T cells in vitro, which increased in the presence of TGF-ß. Interestingly, a further Foxp3(+) Treg expansion was observed if HCs were isolated from regenerated livers. Additionally, the induction of Foxp3(+) Tregs was associated with the Notch signaling pathway, as the ability of HCs to enhance Foxp3 was abolished by γ-secretase inhibition. Furthermore, HC-iTregs showed ability to suppress the proliferative response of CD4(+) T cells to anti-CD3 stimulation in vitro. Thus, HCs may play a pivotal role in the induction of tolerance via Notch-mediated conversion of CD4(+) T cells into Foxp3(+) Tregs upon TCR stimulation.

Hepatocytes contribute to immune regulation in the liver by activation of the Notch signaling pathway in T cells.

The "liver tolerance effect" has been attributed to a unique potential of liver-resident nonprofessional APCs including hepatocytes (HCs) to suppress T cell responses. The exact molecular mechanism of T cell suppression by liver APCs is still largely unknown. In mice, IL-10-dependent T cell suppression is observed after Th1-mediated hepatitis induced by Con A. In this study, we show that HCs, particularly those from regenerating livers of Con A-pretreated mice, induced a regulatory phenotype in naive CD4(+) T cells in vitro. Using reporter mice, we observed that these T regulatory cells released substantial amounts of IL-10, produced IFN-γ, failed to express Foxp3, but suppressed proliferation of responder T cells upon restimulation with anti-CD3 mAb. Hence, these regulatory cells feature a similar phenotype as the recently described IL-10-producing Th1 cells, which are generated upon activation of Notch signaling. Indeed, inhibition of γ-secretase and a disintegrin and metalloproteinase 17 but not a disintegrin and metalloproteinase 10, respectively, which blocked Notch activation, prevented IL-10 secretion. HCs from Con A-pretreated mice showed enhanced expression of the Notch ligand Jagged1 and significantly increased receptor density of Notch1 on CD4(+) T cells. However, HCs from Con A-pretreated IFN regulatory factor 1(-/-) mice, which cannot respond to IFN-γ, as well as those from IFN-γ(-/-) mice failed to augment IL-10 production by CD4(+) T cells. In conclusion, it seems that HCs fine-tune liver inflammation by upregulation of Jagged1 and activation of Notch signaling in Th1 cells. This mechanism might be of particular importance in the regenerating liver subsequent to Th1-mediated hepatitis.

Impaired bone formation and increased osteoclastogenesis in mice lacking chemokine (C-C motif) ligand 5 (Ccl5).

Chemokines play crucial roles in the recruitment of specific hematopoietic cell types, and some of them have been suggested to be involved in the regulation of bone remodeling. Because we have previously observed that chemokine (C-C motif) ligand 2 (Ccl2) and Ccl5 are direct target genes of noncanonical Wnt signaling in osteoblasts, we analyzed the skeletal phenotypes of Ccl2-deficient and Ccl5-deficient mice. In line with previous studies, Ccl2-deficient mice display a moderate reduction of osteoclastogenesis at the age of 6 months. In contrast, 6-month-old Ccl5-deficient mice display osteopenia associated with decreased bone formation and increased osteoclastogenesis. Moreover, unlike in wild-type and Ccl2-deficient mice, large areas of their trabecular and endocortical bone surfaces are not covered by osteoblasts or bone-lining cells, and this is associated with a severe reduction of endosteal bone formation. Although this phenotype diminishes with age, it is important that we could further identify a reduced number of osteal macrophages in 6-month-old Ccl5-deficient mice, because this cell type has previously been reported to promote endosteal bone formation. Because Ccl5-deficient mice also display increased osteoclastogenesis, we finally addressed the question of whether osteal macrophages could differentiate into osteoclasts and/or secrete inhibitors of osteoclastogenesis. For that purpose we isolated these cells by CD11b affinity purification from calvarial cultures and characterized them ex vivo. Here we found that they are unable to differentiate into osteoblasts or osteoclasts, but that their conditioned medium mediates an antiosteoclastogenic effect, possibly caused by interleukin-18 (IL-18), an inhibitor of osteoclastogenesis expressed by osteal macrophages. Taken together, our data provide in vivo evidence supporting the previously suggested role of Ccl5 in bone remodeling. Moreover, to the best of our knowledge, Ccl5-deficient mice represent the first model with a spontaneous partial deficiency of osteal macrophages, a recently identified cell type, whose impact on bone remodeling is just beginning to be understood.

CXCR3 deficiency exacerbates liver disease and abrogates tolerance in a mouse model of immune-mediated hepatitis.

The chemokine receptor CXCR3 is preferentially expressed by Th1 cells and critically involved in their recruitment to inflamed tissue. In a mouse model of immune-mediated liver injury inducible by Con A, we investigated the role of CXCR3 in acute IFN-γ-mediated hepatitis as well as in tolerance induction, which has been shown to depend on IL-10-producing CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Induction of Con A hepatitis resulted in increased intrahepatic expression of the CXCR3 ligands CXCL9, CXCL10, and CXCL11. CXCR3(-/-) mice developed a more severe liver injury with higher plasma transaminase activities and a more pronounced Th1/Th17 response compared with wild-type (wt) animals upon Con A injection. Moreover, CXCR3(-/-) mice did not establish tolerance upon Con A restimulation, although Tregs from CXCR3(-/-) mice were still suppressive in an in vitro suppression assay. Instead, Tregs failed to accumulate in livers of CXCR3(-/-) mice upon Con A restimulation in contrast to those from wt animals. Con A-tolerant wt mice harbored significantly increased numbers of intrahepatic CXCR3(+)T-bet(+) Tregs that produced IL-10 compared with nontolerant animals. IFN-γ deficiency or anti-IFN-γ Ab treatment demonstrated that conversion to CXCR3(+)T-bet(+) Tregs depended on a Th1 response. Accordingly, in an immunotherapeutic approach, CD4(+)CD25(+)Foxp3(+) Tregs from Con A-pretreated CXCR3-deficient mice failed to protect against Con A-induced hepatitis, whereas Tregs from Con A-tolerant wt mice allowed CXCR3-deficient mice to recover from Con A hepatitis. In summary, CXCR3(+)T-bet(+)IL-10(+) Tregs are generated in the liver in dependence of IFN-γ, then disseminated into the organism and specifically migrate into the liver, where they limit immune-mediated liver damage.

Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome.

Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1beta (IL-1beta) by dendritic cells (DCs). The ability of particulates to promote IL-1beta secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1beta secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1beta production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1beta production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigen-specific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b(+)Gr1(-) cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigen-specific cellular immunity.