Lymphotoxin ß receptor activation on macrophages induces cross-tolerance to TLR4 and TLR9 ligands.

Our previous studies indicated that lymphotoxin ß receptor (LTßR) activation controls and downregulates inflammatory reactions. In this study, we report that LTßR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30α, which negatively regulates NF-κB activation induced by TLR signaling. LTßR activation results in a downregulation of proinflammatory cytokine and mediator expression upon TLR restimulation, demonstrating that LTßR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30α-specific small interfering RNA abolished the LTßR-dependent induction of TRIM30α and LTßR-mediated TLR cross-tolerance. Concordantly, LTßR activation on bone marrow-derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Furthermore, we have generated cell type-specific LTßR-deficient mice with ablation of LTßR expression on macrophages/neutrophils (LTßR(flox/flox) × LysM-Cre). In bone marrow-derived macrophages derived from these mice LTßR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTßR expression on macrophages (LTßR(flox/flox) × LysM-Cre) are resistant to LTßR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTßR activation on macrophages by T cell-derived lymphotoxin α(1)ß(2) controls proinflammatory responses by activation of a TRIM30α-controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions.

Neutralization of LIGHT ameliorates acute dextran sodium sulphate-induced intestinal inflammation.

Emerging data indicate that alterations in the expression of tumour necrosis factor (TNF) superfamily members play a crucial role in the pathogenesis of intestinal inflammation. Recent results demonstrated that sustained transgenic expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT; TNFSF14) induced severe intestinal inflammation, suggesting a specific role of LIGHT-mediated signalling to the intestinal compartment. In order to dissect the role of LIGHT in intestinal inflammation, we used LIGHT-deficient mice in the mouse model of acute dextran sodium sulphate-induced colitis. Interestingly, LIGHT-deficient mice were characterized by strongly reduced signs of intestinal inflammation compared with wild-type mice in this experimental model. Determination of mouse LIGHT mRNA expression in colon tissues of wild-type mice revealed a strong induction of mouse LIGHT mRNA expression during acute DSS-induced colitis. We therefore generated anti-mouse LIGHT monoclonal antibodies in LIGHT-deficient mice which bind specifically to LIGHT and are capable of neutralizing the activity of LIGHT in vitro and in vivo. With these antibodies, we demonstrated that neutralization of LIGHT during acute DSS-induced colitis resulted in reduced signs of intestinal inflammation. These data suggest that LIGHT is an important mediator in intestinal inflammation and may serve as a new target for therapeutic intervention.

LTßR expression on hematopoietic cells regulates acute inflammation and influences maturation of myeloid subpopulations.

Lymphotoxin beta-receptor (LTßR) is involved in the formation and maintenance of secondary lymphoid structures, as well as in the regulation of inflammatory responses. Because LTßR lymphoid structure formation continues to develop in infants, we compared two different chimera models: one using adult mice and the other using a transplantation model of neonatal mice. To elucidate the function of LTßR on lymphoid and non-lymphoid cells, we generated bone marrow chimeras on the wild type C57Bl/6 and the LTßR-deficient (LTßR(-/-)) background, and reconstituted the mice with bone marrow cells reciprocally. These chimeric mice were analyzed in the experimental model of acute dextran sulfate sodium-induced colitis. Interestingly, both models revealed not only equal reconstitution levels but also similar immunological responses: LTßR expression on stromal cells is essential for lymph node formation, whereas LTBR on hematopoietic cells is crucial for a decrease in inflammation. In addition, mice lacking LTßR on hematopoietic cells revealed (a) an increase of immature granulocytic cells in the spleen and (b) a reduced proportion of myeloid cells in peripheral blood and spleen expressing CD11b(+)Ly6C(+)Ly6G(-) (myeloid-derived suppressor cells expression profile). In conclusion, LTßR expression on hematopoietic cells seems to be involved in the down-regulation of acute inflammatory reactions paralleled by the appearance of immature myeloid cells.

Lymphotoxin-beta receptor signalling regulates cytokine expression via TRIM30α in a TRAF3-dependent manner.

Our earlier studies indicated that activation of the lymphotoxin beta receptor (LTßR) by T cell derived LTα(1)ß(2) regulates inflammatory cytokine expression. While characterizing the cellular and molecular mechanisms responsible for the down regulation of the inflammatory reaction after LTßR stimulation we were able to identify the specific induction of TRIM30α expression as a result of LTßR signalling in mouse macrophages. Furthermore, we could demonstrate that LTßR activation in these cells results in the down regulation of pro-inflammatory cytokine (e.g. TNF and IL-6) and mediator expression upon TLR4 and TLR9 re-stimulation, demonstrating that LTßR activation on mouse macrophages dampens pro-inflammatory cytokine and mediator expression. Thus, LTßR signalling renders macrophages hypo-responsive to subsequent stimulation with TLR ligands. The observation of an LTßR-mediated TLR-tolerance in the human monocyte cell line THP-1 suggests that similar signalling mechanisms seem to exist in human cells. Signalling pathway analysis clearly demonstrated that LTßR-induced TRIM30α expression is mediated by an IκBα-dependent signalling pathway. Furthermore, the LTßR-induced TRIM30α expression seems to be TRAF3 dependent. Our data suggest that LTßR activation on mouse macrophages is involved in the control of pro-inflammatory cytokine and mediator expression by activation of a signalling pathway that controls exacerbating inflammatory cytokine production.

Lymphotoxin-beta receptor activation on macrophages ameliorates acute DSS-induced intestinal inflammation in a TRIM30α-dependent manner.

Our previous studies indicated that LTßR activation mainly by T cell derived LTα1ß2 is crucial for the control and down-regulation of intestinal inflammation. In order to dissect the cellular and molecular role of LTßR activation in the experimental model of DSS-induced intestinal inflammation, we have generated cell type-specific LTßR-deficient mice with specific ablation of LTßR expression on macrophages/neutrophils (LTßR((flox/flox))×LysM-Cre). These mice develop an exacerbated intestinal inflammation in our experimental model indicating that LTßR expression on macrophages/neutrophils is responsible for the control and down-regulation of the inflammatory reaction. These results were verified by adoptive transfer experiments of BMDM from wild-type and LTßR-deficient mice. Furthermore, transfer of activated CD4+ T cells derived from wild-type mice, but not from LTßR ligand-deficient mice attenuated the signs of intestinal inflammation. Finally, we demonstrate that LTßR activation on BMDM results in induction of TRIM30α, a negative regulator of NFκB activation. Concordantly, ablation of LTßR signaling results in the inability to induce TRIM30α expression concomitant with an increased expression of pro-inflammatory cytokines in our experimental model. Taken together, our data demonstrate that LTßR activation on macrophages by CD4+ T cell derived LTαß controls the pro-inflammatory response by activation of a TRIM30α-dependent signaling pathway, crucial for the down-regulation of the inflammatory response in this experimental model.