Ralph Lucius

Paneth cells as a site of origin for intestinal inflammation

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn’s disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1HM mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn’s disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1ΔIEC) or autophagy function (Atg16l1ΔIEC or Atg7ΔIEC) in intestinal epithelial cells results in each other’s compensatory engagement, and severe spontaneous Crohn’s-disease-like transmural ileitis if both mechanisms are compromised. Xbp1ΔIEC mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn’s disease as a specific disorder of Paneth cells.

Activation of microglia by human neuromelanin is NF-κB dependent and involves p38 mitogen-activated protein kinase: implications for Parkinson’s disease

It has been suggested that microglial inflammation augments the progression of Parkinsons disease (PD). However, endogenous factors initiating microglial activation are largely unknown. We therefore investigated the effects of human neuromelanin (NM) on the release of neurotoxic mediators and the underlying signaling pathways from rat microglia in vitro. The addition of NM to microglial cultures induced positive chemotactic effects, activated the proinflammatory transcription factor nuclear factor κB (NF‐κB) via phosphorylation and degradation of the inhibitor protein κB (IκB), and led to an up‐regulation of tumor necrosis factor α, interleukin‐6, and nitric oxide. The impairment of NF‐κB function by the IκB kinase inhibitor sulfasalazine was paralleled by a decline in neurotoxic mediators. NM also activated p38 mitogen‐activated protein kinase (MAPK), the inhibition of this pathway by SB203580 diminished phosphorylation of the transactivation domain of the p65 subunit of NF‐κB. These findings demonstrate a crucial role of NM in the pathogenesis of PD by augmentation of microglial activation, leading to a vicious cycle of neuronal death, exposure of additional neuromelanin, and chronification of inflammation. The antagonization of microglial activation by a pharmacological intervention targeting microglial NF‐κB or p38 MAPK could point to additional venues in the treatment of PD.

c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia.

The activation and function of c‐Jun N‐terminal kinases (JNKs) were investigated in primary microglia cultures from neonatal rat brain, which express all three JNK isoforms. Lipopolysaccharide (LPS), tumor necrosis factor‐α (TNF‐α), and thrombin preparations induced a rapid and lasting activation of JNKs in the cytoplasm. In the nucleus, the activation patterns were rather complex. In untreated microglia, the small pool of nuclear JNKs was strongly activated, while the high‐affinity JNK substrate c‐Jun was only weakly phosphorylated. Stimulation with LPS increased the total amount of nuclear JNKs and the phosphorylation of the transcription factor c‐Jun. Levels of activated JNKs in the nucleus, however, rapidly decreased. Analysis of the nuclear JNK isoforms revealed that the amount of JNK1 declined, while JNK2 increased, and the weakly expressed JNK3 did not vary. This observation suggests that JNK2 is mainly responsible for the activation of c‐Jun in this context. Upstream of JNKs, LPS induced a lasting activation of the constitutively present JNK kinase MKK4. The function of JNKs in LPS‐triggered cellular reactions was investigated using SP600125 (0.5–5 μM), a direct inhibitor of JNKs. Inhibition of JNKs reduced the LPS‐induced metabolic activity and induction of the AP‐1 target genes cyclooxygenase‐2 (Cox‐2), TNF‐α, monocyte chemoattractant protein‐1 (MCP‐1), and interleukin‐6 (IL‐6) in response to LPS, while ERK1/2 and p38α had a more pronounced effect on LPS‐induced cellular enlargement than JNKs. In summary, JNKs are essential mediators of relevant pro‐inflammatory functions in microglia with different contributions of the JNK isoforms.

Dimethylfumarate inhibits microglial and astrocytic inflammation by suppressing the synthesis of nitric oxide, IL-1β, TNF-α and IL-6 in an in-vitro model of brain inflammation

BackgroundBrain inflammation plays a central role in multiple sclerosis (MS). Dimethylfumarate (DMF), the main ingredient of an oral formulation of fumaric acid esters with proven therapeutic efficacy in psoriasis, has recently been found to ameliorate the course of relapsing-remitting MS. Glial cells are the effector cells of neuroinflammation; however, little is known of the effect of DMF on microglia and astrocytes. The purpose of this study was to use an established in vitro model of brain inflammation to determine if DMF modulates the release of neurotoxic molecules from microglia and astrocytes, thus inhibiting glial inflammation.MethodsPrimary microglial and astrocytic cell cultures were prepared from cerebral cortices of neonatal rats. The control cells were treated with LPS, an accepted inducer of pro-inflammatory properties in glial cells, and the experimental groups with LPS and DMF in different concentrations. After stimulation/incubation, the generation of nitric oxide (NO) in the cell culture supernatants was determined by measuring nitrite accumulation in the medium using Griess reagent. After 6 hours of treatment RT-PCR was used to determine transcription levels of iNOS, IL-1β, IL-6 and TNF-α mRNA in microglial and astrocytic cell cultures initially treated with DMF, followed after 30 min by LPS treatment. Moreover, we investigated possible involvement of the ERK and Nrf-2 transduction pathway in microglia using western blot analysis.ResultsPretreatment with DMF decreased synthesis of the proinflammatory mediators iNOS, TNF-α, IL-1β and IL-6 at the RNA level in activated microglia and astrocytes in vitro, associated with a decrease in ERK phosphorylation in microglia.ConclusionsCollectively, these results suggest that the neuroprotective effects of DMF may be in part functionally attributable to the compounds ability to inhibit expression of multiple neuroinflammatory mediators in brain of MS patients.

Vascular endothelial growth factor induces chemotaxis and proliferation of microglial cells

Vascular endothelial growth factor (VEGF) is an angiogenic peptide that is produced in the brain after ischemia, injury or in malignant gliomas. Since these pathological conditions are associated with the infiltration of microglial cells, we investigated the expression of VEGF receptors (VEGFR) and possible effects of VEGF on cultivated microglial cells. As shown by reverse transcription-polymerase chain reaction and immunocytochemistry, rat microglial cells as well as the murine cell line BV-2 express the VEGFR-1, but not VEGFR-2. Murine VEGF induced 3H-thymidine incorporation into DNA of murine and rat microglial cells as well as chemotaxis in Boyden chamber assays. However, VEGF did not alter the phosphorylation of mitogen-activated protein kinases and only slightly that of the kinase Akt. These results show that microglial cells are targets for VEGF which induces migration and proliferation of these immunocompetent cells in the brain.

Angiotensin II accelerates functional recovery in the rat sciatic nerve in vivo: role of the AT2 receptor and the transcription factor NF-κB

The AT2 receptor regulates several functions of nerve cells, e.g., ionic fluxes, cell differentiation, and axonal regeneration, but also modulates programmed cell death. We tested the hypothesis that angiotensin II (ANG II) via its AT2 receptor not only promotes regeneration but also functional recovery after sciatic nerve crush in adult rats. ANG II (10−7, 10−9, 10−11 M) applied locally via osmotic minipumps promoted functional recovery with maximal effects after the lowest concentration. The toe spread distance as a parameter for re‐innervation after 20 days was significantly (P<0.01) greater (10.2±10.27 mm) compared with the control group (8.73±0.16 mm). The response to local electrical stimulation (return of sensorimotor function) was reduced to 14.6 days vs. 17.9 days in the control group (P<0.01). The AT2 receptor antagonist PD 123319 administered alone or in combination with ANG II completely prevented the ANG II‐induced recovery, whereas the AT1 receptor antagonist losartan had no effect. Furthermore, ANG II induces, via the AT2 receptor, activation of the transcription factor NF‐κB in Schwann cells. Histological criteria, morphometric analyses, and electron microscopy confirmed the functional data. These results are the first to present direct evidence for an involvement of the AT2 receptor and NF‐κB in peripheral nerve regeneration.

Involvement of benzodiazepine receptors in neuroinflammatory and neurodegenerative diseases: evidence from activated microglial cells in vitro

Increased binding of a ligand for the peripheral benzodiazepine binding receptor is currently used in PET studies as an in vivo measurement of inflammation in diseases like multiple sclerosis and Alzheimers disease. Although peripheral-type benzodiazepin receptors (PBRs) are abundant in many cell types and expressed in the CNS physiologically only at low levels, previous reports suggest that after experimental lesions in animal models and in human neurodegenerative/-inflammatory diseases upregulated PBR expression with increased binding of its ligand PK11195 is confined mainly to activated microglia in vivo/in situ. Because the functional role of the PBR is unknown, we confirm by immunohistochemistry and PCR (I) that this receptor is expressed on microglia in vitro and (II) that benzodiazepines modulate proliferation of microglial cells and the release of the inflammatory molecules nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in cell culture supernatants of primary rat microglia. Compared to lipopolysaccharide-activated controls the release of NO was markedly decreased in cultures treated with benzodiazepines (clonazepam, midazolam, diazepam) and the PBR ligand PK11195. Moreover, release of TNF-alpha and proliferation was significantly inhibited in the benzodiazepine-treated groups. These findings link the in vivo data of elevated PBR levels in neurodegenerative/-inflammatory diseases to a functional role and opens up possible therapeutic intervention targeting the PBR in microglia.

Intrathecal synthesis of monocyte chemoattractant protein-1 (MCP-1) in amyotrophic lateral sclerosis: further evidence for microglial activation in neurodegeneration.

Autopsy studies and animal experiments suggest that microglial inflammation contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS). Monocyte-chemoattractant protein (MCP-1) might play an important role in microglial recruitment. We studied MCP-1 levels in sera and cerebrospinal fluid of 29 ALS patients and compared the results with 11 control patients with tension headache. The MCP-1 level was determined using enzyme-linked immunosorbent assays (ELISA). A significant increase in cerebrospinal fluid MCP-1 level but not serum level was seen in the patients with ALS compared to the control subjects. These results suggest that cerebrospinal fluid MCP-1 activity may be a sensitive marker for neuroinflammation in ALS useful for monitoring treatment trials in ALS.

Sulforaphane suppresses LPS-induced inflammation in primary rat microglia

Objective and designThe aim of this study was to investigate the signal transduction pathways involved in sulforaphane (SF) mediated inhibition of the inflammatory response to lipopolysaccharide (LPS). Additionally, we investigated the effects of SF and LPS on the activity of Nrf2.MaterialPrimary rat microglia and the murine microglia cell line BV2 were used.TreatmentCells were treated with LPS with or without SF.MethodsCell viability was measured via WST-assay. Real-time RT-PCR was performed to analyze cytokine mRNA levels. The nitric oxide (NO) release was measured in LPS-stimulated microglia. The induction of various signal transduction pathways and Nrf2 was determined by Western blotting. NF-κB and AP-1 activation was measured by dual luciferase assay.ResultsWe showed that SF attenuates the LPS-induced increase of IL-1β, IL-6, and TNF-α expression in microglia. In addition, SF significantly decreases the NO in a concentration-dependent manner. SF inhibits LPS-stimulated ERK1/2 and JNK phosphorylation and thereby inhibits the LPS-induced activation of NF-κB- and activator protein-1 (AP-1). Moreover, SF and LPS together are able to induce Nrf2 activation.ConclusionsWe showed that SF, and also LPS by itself, are able to activate the cell’s defence against oxidative and electrophilic stress. We conclude that SF could be a candidate agent for anti-inflammatory treatment of the central nervous system.

Force-Dependent Development of Neuropathic Central Pain and Time-Related CCL2/CCR2 Expression after Graded Spinal Cord Contusion Injuries of the Rat

Spinal cord injury (SCI) often results in intractable chronic central pain syndromes. Recently chemokines such as CCL2 were identified as possible key integrators of neuropathic pain and inflammation after peripheral nerve lesion. The focus of the current study was the investigation of time-dependent CCL2 and CCR2 expression in relation to central neuropathic pain development after spinal cord impact lesions of 100, 150, or 200 kdyn force on spinal cord level T9 in adult rats. Below-level pain was monitored with weekly sensory testing for 42 days after SCI. In parallel expression of CCL2/CCR2 on cervical, thoracic, and lumbar levels was investigated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry early (7 days [7d]), intermediate (15d), and late (42d) after lesion. Cellular source and anatomical pain related expression was determined by double-immunohistochemistry. Force-defined SCI led to acute mechanical hypersensitivity in all lesion groups, and to persistent below-level pain in severely injured animals. While in the early post-operative time course, CCL2 and CCR2 were expressed in astroglia and granulocytes only on level T9; there was additional astroglial CCL2 expression in dorsal columns and dorsal horns above and below T9 of severely injured animals 42d after lesion. In dorsal horns (level L3-L5) of animals exhibiting chronic below-level pain CCL2 was co-expressed with transmitters and receptors that are involved in nociceptive processing like calcitonin gene-related peptide (CGRP), Substance-P, vanilloid-receptor-1, and its activated phosphorylated form. These data demonstrate lesion grade dependence of below-level pain development and suggest chemokines as potential candidates for integrating inflammation and central neuropathic pain after SCI.