Joris Vanderlocht

Myeloid Type I Interferon Signaling Promotes Atherosclerosis by Stimulating Macrophage Recruitment to Lesions

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Localization of the lipopolysaccharide recognition complex in the human healthy and inflamed premature and adult gut

Background: Microbiota in the intestinal lumen provide an abundant source of potentially detrimental antigens, including lipopolysaccharide (LPS), a potent immunostimulatory product of Gram‐negative bacteria recognized by the host via TLR‐4 and MD‐2. An aberrant immune response to LPS or other bacterial antigens has been linked to inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). Methods: We investigated which cells express MD‐2 in the normal and inflamed ileum from neonates and adults by immunohistochemistry. Moreover, MD‐2 and TLR4 mRNA expression in normal adult ileum was studied by reverse‐transcription polymerase chain reaction (RT‐PCR) on cells isolated by laser capture microdissection. Results: Premature infants did not show MD‐2 expression either in epithelial cells or in the lamina propria. Similarly, MD‐2 was absent in epithelial cells and lamina propria inflammatory cells in preterm infants with NEC. MD‐2 protein in the healthy term neonatal and adult ileum was predominantly expressed by Paneth cells and some resident inflammatory cells in the lamina propria. MD‐2 and TLR‐4 mRNA expression was restricted to crypt cells. Also in IBD, Paneth cells were still the sole MD‐2‐expressing epithelial cells, whereas inflammatory cells (mainly plasma cells) were responsible for the vast majority of the MD‐2 expression. Conclusions: The absence of MD‐2 in the immature neonatal gut suggests impaired LPS sensing, which could predispose neonates to NEC upon microbial colonization of the immature intestine. The apparent expression of MD‐2 by Paneth cells supports the critical concept that these cells respond to luminal bacterial products in order to maintain homeostasis with the intestinal microbiota in vivo. (Inflamm Bowel Dis 2010;)

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106 MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Cerebral inflammation and mobilization of the peripheral immune system following global hypoxia-ischemia in preterm sheep

BackgroundHypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI.MethodsPreterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis.ResultsGlobal HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function.ConclusionsOur data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.

Follicular T helper cells and humoral reactivity in kidney transplant patients

Memory B cells play a pivotal role in alloreactivity in kidney transplantation. Follicular T helper (Tfh) cells play an important role in the differentiation of B cells into immunoglobulin‐producing plasmablasts [through interleukin (IL)‐21]. It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney transplant patients, therefore we investigated the absolute numbers and function of peripheral Tfh cells (CD4POSCXCR5POS T cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor‐specific anti‐human leucocyte antigen (HLA) antibodies (DSA), and the presence of Tfh cells in rejection biopsies. After transplantation peripheral Tfh cell numbers remained stable, while their IL‐21‐producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral Tfh cells still had the capacity to induce B cell differentiation and immunoglobulin production, which could be inhibited by an IL‐21‐receptor‐antagonist. After transplantation the quantity of Tfh cells was the highest in patients with pre‐existent DSA. In kidney biopsies taken during rejection, Tfh cells co‐localized with B cells and immunoglobulins in follicular‐like structures. Our data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu.

Inflammation restraining effects of prostaglandin E2 on natural killer–dendritic cell (NK-DC) interaction are imprinted during DC maturation

Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood-derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44(+) NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.

In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT.

Natural Killer Cells: The Secret Weapon in Dendritic Cell Vaccination Strategies

In cancer therapy, dendritic cell (DC) vaccination is still being explored. Clinical responses, however, are diverse and there is a lack of immunologic readout systems that correspond with clinical outcome. Only in the minority of patients, T-cell responses correlate with clinical outcome, indicating that other immune cells also gain anticancer activity. We still have limited knowledge of the effect of DC vaccination on different immune effector cells. However, it has been shown that bidirectional cross-talk between natural killer (NK) cells and DCs is responsible for enhanced activation of both cell types and increases their antitumor activity. In this review, we postulate the possibility that NK cells are the secret weapons in DC vaccination and studying their behavior together with T-cell activation in vaccinated individuals might predict clinical outcome. Clin Cancer Res; 20(5); 1095–103. ©2014 AACR.

Klebsiella pneumoniae-triggered DC recruit human NK cells in a CCR5-dependent manner leading to increased CCL19-responsiveness and activation of NK cells.

Besides their role in destruction of altered self‐cells, NK cells have been shown to potentiate T‐cell responses by interacting with DC. To take advantage of NK–DC crosstalk in therapeutic DC‐based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK‐cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5‐dependent manner. An additional mechanism of DC‐induced NK‐cell recruitment is characterized by the induction of CCR7 expression on CD56dimCD16+ NK cells after physical contact with membrane fraction of K. pneumoniae‐matured DC, resulting in an enhanced migratory responsiveness to the lymph node‐associated chemokine CCL19. Bacterial fragment‐matured DC do not only mediate NK‐cell migration but also meet the prerequisites needed for augmentation of NK‐cell cytotoxicity and IFN‐γ production, the latter of which contributes to Th1 polarization.

INCREASED TUMOR-SPECIFIC CD8(+) T CELL INDUCTION BY DENDRITIC CELLS MATURED WITH A CLINICAL GRADE TLR-AGONIST IN COMBINATION WITH IFN-gamma

The limited response rate of cancer patients treated with dendritic cell (DC)-based vaccines indicates that vast improvements remain necessary. In many murine tumour models it has been demonstrated that the use of innate triggers (e.g. TLR triggers) in the maturation of DC results in higher efficacy. However, as few of these innate triggers are generated clinical grade, there remains a great necessity to fill the gap between fundamental mouse studies and a clinical trial in humans. In the present study we used a TLR2/4-agonist (FMKp which is available clinical grade) in combination with IFN-γ (FI-cocktail) in the maturation of elutriated monocyte-derived DC and compared it with the most used DC in current clinical trials (TNF-α/PGE-2, i.e. TP-cocktail). In addition to the assessment of CD4+ T cell polarizing capacity, we compared the quantity and intrinsic quality of induced CD8+ T cells of 2 different DC maturation protocols with all cells from the same donor. Besides differences in the cytokine profile, which could be coupled to increased Th1 and Th17 polarization, we demonstrate in this study that FMKp/IFN-γ matured DC are twice as effective in inducing cytotoxic T cells against known tumor antigens. Both DCs induced phenotypically equivalent effector memory CD8+ T cells that did not show a significant difference in their intrinsic capacity to kill tumor cells. These findings point to the therapeutic applicability of FI-DC as superior inducers of functional antigen-specific T cells. Their increased chemokine secretion is suggestive of a mechanism by which these DC may compensate for the limited migration observed for all ex vivo cultured DC when applied in patients.