Cobi J. Heijnen

Ventilator–induced lung injury (VILI) is characterized by vascular leakage and inflammatory responses eventually leading to pulmonary dysfunction. Vascular endothelial growth factor (VEGF) has been proposed to be involved in the pathogenesis of VILI. This study examines the inhibitory effect of dexamethasone on VEGF expression, inflammation and alveolar–capillary barrier dysfunction in an established murine model of VILI. Healthy male C57Bl/6 mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with an inspiratory pressure of 10 cmH₂O (“lower” tidal volumes of ∼7.5 ml/kg; LV_T) or 18 cmH₂O (“higher” tidal volumes of ∼15 ml/kg; HV_T). Dexamethasone was intravenously administered at the initiation of HV_T–ventilation. Non–ventilated mice served as controls. Study endpoints included VEGF and inflammatory mediator expression in lung tissue, neutrophil and protein levels in bronchoalveolar lavage fluid, PaO₂ to FiO₂ ratios and lung wet to dry ratios. Particularly HV_T–ventilation led to alveolar–capillary barrier dysfunction as reflected by reduced PaO₂ to FiO₂ ratios, elevated alveolar protein levels and increased lung wet to dry ratios. Moreover, VILI was associated with enhanced VEGF production, inflammatory mediator expression and neutrophil infiltration. Dexamethasone treatment inhibited VEGF and pro–inflammatory response in lungs of HV_T–ventilated mice, without improving alveolar–capillary permeability, gas exchange and pulmonary edema formation. Dexamethasone treatment completely abolishes ventilator–induced VEGF expression and inflammation. However, dexamethasone does not protect against alveolar–capillary barrier dysfunction in an established murine model of VILI.

Scientists and clinicians have long used the level of hormones or the level of inflammatory mediators as an important parameter of functional activity of the neuro-endocrine or immune system. However, not much focus has been given to the role of the sensitivity of the target tissue, such as receptors or components of the intracellular signalosome. This is important because, as recent literature has shown, the sensitivity of the target tissue may change during pathological processes such as inflammation or chronic stress. This chapter focuses on changes in sensitivity of target tissue by focussing on a kinase known as G protein receptor kinase 2 (GRK2), which appears to be an important regulator of the severity and duration of inflammatory pain. GRK2 regulates the sensitivity of target systems, including immune and nervous systems, for signals given by G protein-coupled receptors via regulating receptor desensitization. In addition, GRK2 regulates intracellular signaling via direct effects on elements of the signalosome of the cell. The contribution of GRK2 to chronic pain is discussed in the context of the idea that regulation of the sensitivity of target systems is an important process during neuro-inflammation and should be taken into account when investigating neuro-immune communication in inflammatory pathologies.

Mesial temporal lobe epilepsy (mTLE) is a chronic and often treatment-refractory brain disorder characterized by recurrent seizures originating from the hippocampus. The pathogenic mechanisms underlying mTLE remain largely unknown. Recent clinical and experimental evidence supports a role of various inflammatory mediators in mTLE. Here, we performed protein expression profiling of 40 inflammatory mediators in surgical resection material from mTLE patients with and without hippocampal sclerosis, and autopsy controls using a multiplex bead-based immunoassay. In mTLE patients we identified 21 upregulated inflammatory mediators, including 10 cytokines and 7 chemokines. Many of these upregulated mediators have not previously been implicated in mTLE (for example, CCL22, IL-7 and IL-25). Comparing the three patient groups, two main hippocampal expression patterns could be distinguished, pattern I (for example, IL-10 and IL-25) showing increased expression in mTLE + HS patients compared to mTLE-HS and controls, and pattern II (for example, CCL4 and IL-7) showing increased expression in both mTLE groups compared to controls. Upregulation of a subset of inflammatory mediators (for example, IL-25 and IL-7) could not only be detected in the hippocampus of mTLE patients, but also in the neocortex. Principle component analysis was used to cluster the inflammatory mediators into several components. Follow-up analyses of the identified components revealed that the three patient groups could be discriminated based on their unique expression profiles. Immunocytochemistry showed that IL-25 IR (pattern I) and CCL4 IR (pattern II) were localized in astrocytes and microglia, whereas IL-25 IR was also detected in neurons. Our data shows co-activation of multiple inflammatory mediators in hippocampus and neocortex of mTLE patients, indicating activation of multiple pro- and anti-epileptogenic immune pathways in this disease.

It has been suggested that pro-inflammatory cytokine signaling to the brain may contribute to severe fatigue. We propose that not only the level of circulating cytokines, but also increased reactivity of target cells to cytokines contributes to the effect of cytokines on behavior. Based on this concept, we assessed the reactivity of peripheral blood cells to IL-1β in vitro as a novel approach to investigate whether severe fatigue is associated with increased pro-inflammatory signaling.

The aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women. Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001). H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM.

Background: Chronic pain is often associated with microglia activation in the spinal cord. We recently showed that microglial levels of the kinase G protein–coupled receptor kinase (GRK)2 are reduced in models of chronic pain. We also found that mice with a cell-specific reduction of around 50% in GRK2 level in microglia/macrophages (LysM-GRK2^+/− mice) develop prolonged inflammatory hyperalgesia concomitantly with ongoing spinal microglia/macrophage activation. The microRNA miR-124 is thought to keep microglia/macrophages in brain and spinal cord in a quiescent state. In the present study, we investigated the contribution of miR-124 to regulation of hyperalgesia and microglia/macrophage activation in GRK2-deficient mice. In addition, we investigated the effect of miR-124 on chronic inflammatory and neuropathic pain in wild-type (WT) mice. Methods: Hyperalgesia was induced by intraplantar IL-1β in WT and LysM-GRK2^+/− mice. We determined spinal cord microglia/macrophage miR-124 expression and levels of pro-inflammatory M1 and anti-inflammatory M2 activation markers. The effect of intrathecal miR-124 treatment on IL-1β-induced hyperalgesia and spinal M1/M2 phenotype, and on carrageenan-induced and spared nerve injury-induced chronic hyperalgesia in WT mice was analyzed. Results: Transition from acute to persistent hyperalgesia in LysM-GRK2^+/− mice is associated with reduced spinal cord microglia miR-124 levels. In our LysM-GRK2^+/− mice, there was a switch towards a pro-inflammatory M1 phenotype together with increased pro-inflammatory cytokine production. Intrathecal administration of miR-124 completely prevented the transition to persistent pain in response to IL-1β in LysM-GRK2^+/− mice. The miR-124 treatment also normalized expression of spinal M1/M2 markers of LysM-GRK2^+/− mice. Moreover, intrathecal miR-124 treatment reversed the persistent hyperalgesia induced by carrageenan in WT mice and prevented development of mechanical allodynia in the spared nerve injury model of chronic neuropathic pain in WT mice. Conclusions: We present the first evidence that intrathecal miR-124 treatment can be used to prevent and treat persistent inflammatory and neuropathic pain. In addition, we show for the first time that persistent hyperalgesia in GRK2-deficient mice is associated with an increased ratio of M1/M2 type markers in spinal cord microglia/macrophages, which is restored by miR-124 treatment. We propose that intrathecal miR-124 treatment might be a powerful novel treatment for pathological chronic pain with persistent microglia activation.