Karin Koekkoek

Prolongation of skin graft survival by modulation of the alloimmune response with alternatively activated dendritic cells.

Background. Activation of immature dendritic cells (DC) in the presence of the glucocorticoid hormone dexamethasone (DEX) results in alternatively matured DC that present antigen in the absence of a proper co-stimulatory context. This maturation process is irreversible, making these cells an attractive potential tool for the induction of antigen-specific T-cell tolerance in vivo. The authors explored the possibility of using these DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. Methods. Immature dendritic cells (D1, an immature splenic DC line derived from B6 mice) were pretreated with DEX for 24 hr, after which lipopolysaccharide or nothing was added to the culture for another 48 hr. These cells were analyzed for their in vitro and in vivo stimulating or tolerizing capacities. Results. In line with their phenotype, including decreased interleukin (IL)-12 production, in vitro co-culture of alternatively matured D1 (B6 origin; H-2b) with completely allogeneic T cells of BALB/c origin led to a significant decrease in the alloreactive T-cell response. A single injection of 1×106 alternatively matured H-2b DC into BALB/c mice induced a different alloimmune response compared with mature DC. The responding T cells showed a lower proliferation rate and a lower interferon-&ggr; production, whereas a significantly higher proportion of the cells produced IL-10 as measured ex vivo by enzyme-linked immunospot assay. Furthermore, injection with alternatively matured DC, followed by transplantation of fully mismatched skin grafts (C57BL/6), led to a significantly prolonged survival compared with that of mature DC-pretreated mice or untreated mice. The immunomodulatory effect was antigen specific, as third-party reactive alloresponses were not affected. Conclusions. The authors’ data constitute the first direct demonstration that DC alternatively matured in the presence of glucocorticoid hormones can be exploited for the specific suppression of the alloreactive Th1 response, resulting in a delayed skin graft rejection in a complete major histocompatibility complex-incompatible strain combination.

Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages.

Macrophages have been demonstrated to suppress T cell responses by producing reactive oxygen species (ROS) leading to the subsequent induction of T regulatory cells in a ROS-dependent manner. Macrophages may therefore be instrumental in downregulating T cell responses in situations of exacerbated immune responses. Here we investigated the effect of immunosuppressive drugs on ROS production by macrophage subsets and the subsequent effects on T cell activation. Macrophage types 1 and 2 were differentiated with GM-CSF or M-CSF, in presence or absence of dexamethasone, cyclosporine A, FK506, rapamycin, or mycophenolic acid. The ROS producing capacity of fully differentiated Mph was highest in anti-inflammatory Mph2 and not affected by exposure to immunosuppressive drugs. However, presence of rapamycin during Mph2 differentiation decreased the ROS production of these cells. In contrast, other immunosuppressive drugs, with dexamethasone being the most potent, increased the ROS producing capacity of Mph2. Intriguingly although the ROS producing ability of Mph1 was unaffected, dexamethasone strongly increased the ROS producing capabilities of dendritic cells. Both at the mRNA and protein level we found that dexamethasone enhanced the expression of NOX2 protein p47(phox). Functionally, dexamethasone further enhanced the capacity of Mph2 to suppress T cell mediated IFN-γ and IL-4 production. In vivo, only in rats with normal ROS production (congenic DA.Ncf1(E3/E3)) it was observed that dexamethasone injection resulted in long-lasting upregulation of ROS production by macrophages and induced higher levels of Treg in a ROS-dependent manner. In conclusion, we show that the anti-inflammatory drug dexamethasone increases the ROS producing capacity of macrophages.

Differentially modulated dendritic cells induce regulatory T cells with different characteristics

Dexamethason (DEX) treated DC display several features that establish them as candidates for specific allogeneic tolerance induction. We report the results of in vitro studies of polarization of the alloimmune T cell response with two types of differentially modulated human DC. Both DEX treated DC triggered by LPS for 6 h (DEX6-DC) and DEX treated DC triggered by LPS for 48 h (DEX48-DC) acquired low levels of costimulatory, adhesion, and MHC class II molecules compared with mature DC (mDC). In contrast to mDC, both DEX6-DC and DEX48-DC did not produce any IL-12. DEX6-DC were able to produce significant amounts of IL-10 whereas DEX48-DC did not actively produce IL-10. Conversely, the induction of IL-10 producing cells was significantly increased when PBL were stimulated with DEX48-DC compared with DEX6-DC. Both stimulation of PBL with DEX6-DC and DEX48-DC led to the induction of cell populations able to suppress the proliferative alloimmune response of primed T cells in a cell-cell contact independent and antigen-nonspecific manner. Tregs obtained after stimulation with DEX48-DC were also able to inhibit the IFN-gamma production of the effector cells and this effect could be blocked by anti-IL-10. Tregs induced by DEX6-DC produced similar amounts of IL-10, yet were not able to inhibit IFN-gamma production of the effector T cells, indicating a different mechanism. In summary, we show that differential modulation of DC results in the induction of different populations of regulatory T cells.

Subsets of human type 2 macrophages show differential capacity to produce reactive oxygen species

Reactive oxygen species (ROS) produced by macrophages have recently been shown to have immunosuppressive properties and induce regulatory T cells. Here we investigated the ROS producing capacity of well-defined human Mph2 subsets and studied the contribution of ROS in the Mph-T cell interaction. Mph were generated from monocytes using M-CSF (Mph2), IL-4 (Mph2a), or IL-10 (Mph2c). Upon PMA stimulation, Mph2 and Mph2c showed a high ROS producing capacity, whereas this was low for Mph2a. Mph2 and Mph2c displayed a reduced T cell stimulatory capacity compared to Mph2a. Addition of the ROS inhibitor DPI decreased the T cell proliferation and IFN-γ production. When testing directly on Mph, DPI dose-dependently decreased the IL-10 and IL-12p40 production of CD40L-stimulated Mph2 subsets. In conclusion, the ROS producing capacity is different among human Mph type-2 subsets. In all cases, DPI suppressed T cell proliferation and cytokine production, indicating a ROS-dependent mechanism of T cell activation.

The NOX2-mediated ROS producing capacity of recipient cells is associated with reduced T cell infiltrate in an experimental model of chronic renal allograft inflammation

We previously showed that anti-inflammatory Mph (Mph2) can both in vitro and in vivo induce regulatory T cells (Tregs) in a reactive oxygen species (ROS)-dependent fashion. As influx of Mph is an important characteristic of chronic inflammatory responses, we investigated the impact of NOX2-mediated ROS production by recipient cells in an experimental model of chronic allograft inflammation. We used a kidney transplantation (Tx) model with Lewis (Lew) rats as donor and congenic DA.Ncf1(DA/DA) (low ROS) and DA.Ncf1(E3/E3) (normal ROS) rats as recipients. At day 7 the contralateral kidney was removed, and the animals were sacrificed four weeks after Tx. Renal function and injury were monitored in serum and urine and the composition of the infiltrate was analyzed by immunohistochemistry. Four weeks after Tx, large leukocyte clusters were observed in the allograft, in which signs of ROS production could be demonstrated. These clusters showed no difference regarding composition of myeloid cells or the number of FoxP3 positive cells. However, T cell infiltrate was significantly reduced in the DA.Ncf1(E3/E3) recipients having normal ROS production. Therefore, this study suggests a regulatory effect of ROS on T cell infiltration, but no effect on other inflammatory cells in the allograft.

Chronic kidney failure mineral bone disorder leads to a permanent loss of hematopoietic stem cells through dysfunction of the stem cell niche

In chronic kidney disease (CKD), endothelial injury, is associated with disease progression and an increased risk for cardiovascular complications. Circulating cells with vascular reparative functions are hematopoietic and also reduced in CKD. To explore the mechanistic basis behind these observations, we have investigated hematopoietic stem cell (HSC) homeostasis in a mouse model for non-progressive CKD-mineral and bone disorder with experimentally induced chronic renal failure (CRF). In mice subjected to 12 weeks of CRF, bone marrow HSC frequencies were decreased and transplantation of bone marrow cells from CRF donors showed a decrease in long-term HSC repopulation compared to controls. This loss was directly associated with a CRF-induced defect in the HSC niche affecting the cell cycle status of HSC and could not be restored by the PTH-reducing agent cinacalcet. In CRF, frequencies of quiescent (G0) HSC were decreased coinciding with an increase in hematopoietic progenitor cells (HPC) in the S-and G2-phases of cell cycle. Moreover, in CRF mice, HSC-niche supporting macrophages were decreased compared to controls concomitant to impaired B lymphopoiesis. Our data point to a permanent loss of HSC and may provide insight into the root cause of the loss of homeostatic potential in CKD.