Inflammatory dysregulation of monocytes in pediatric patients with obsessive-compulsive disorder

Background

Although the exact etiology of obsessive-compulsive disorder (OCD) is unknown, there is growing evidence of a role for immune dysregulation in the pathophysiology of the disease, especially in the innate immune system including the microglia. To test this hypothesis, we studied inflammatory markers in monocytes from pediatric patients with OCD and from healthy controls.

Methods

We determined the percentages of total monocytes, CD16+ monocytes, and classical (CD14^highCD16?), intermediate (CD14^highCD16^low), and non-classical (CD14^lowCD16^high) monocyte subsets in 102 patients with early-onset OCD and in 47 healthy controls. Moreover, proinflammatory cytokine production (GM-CSF, IL-1β, IL-6, IL-8, and TNF-α) was measured by multiplex Luminex analysis in isolated monocyte cultures, in basal conditions, after exposure to lipopolysaccharide (LPS) to stimulate immune response or after exposure to LPS and the immunosuppressant dexamethasone.

Results

OCD patients had significantly higher percentages of total monocytes and CD16+ monocytes than healthy controls, mainly due to an increase in the intermediate subset but also in the non-classical monocytes. Monocytes from OCD patients released higher amounts of GM-CSF, IL-1β, IL-6, IL-8, and TNF-α than healthy controls after exposure to LPS. However, there were no significant differences in basal cytokine production or the sensitivity of monocytes to dexamethasone treatment between both groups. Based on monocyte subset distribution and cytokine production after LPS stimulation, patients receiving psychoactive medications seem to have an intermediate inflammatory profile, that is, lower than non-medicated OCD individuals and higher than healthy controls.

Conclusions

These results strongly support the involvement of an enhanced proinflammatory innate immune response in the etiopathogenesis of early-onset OCD.

     

Deficiencies in elements involved in TLR4-receptor signalling in RBL-2H3 cells

Objective

RBL-2H3 cells express Toll-like receptors, including TLR4. This study aims to assess various aspects of the TLR4 pathway.

Methods

RBL-2H3 cells were indirectly stained for cell surface TLR4, 25 CD14 and intracellular MyD88 proteins and analysed through flow cytometry for single-colour staining.

Results

While TLR4-receptors are expressed in RBL-2H3 cells, associated elements involved in the signaling pathway, CD14 and MyD88, are not.

Conclusion

Care should be taken if RBL-2H3 cells are used to study aspects of the innate immune system in mast cells.

     

TLR4 regulates insulin-resistant proteins to increase apoptosis in the mouse retina

Objective and design

Work in multiple organs has suggested that toll-like receptor 4 (TLR4) may play a role in insulin resistance. Additional studies have shown a negative role for TLR4 on retinal health. We have previously reported that β-adrenergic receptors can regulate both TLR4 signal transduction, as well as insulin signaling in the retina and in retinal endothelial cells. Thus, we hypothesized that TLR4 would regulate retinal insulin signaling.

Materials and methods

We used endothelial cell-specific TLR4 knockout mice, as well as TLR4-overexpressing mice for these studies.

Methods

Western blotting and ELISA analyses were done for investigations of insulin receptor, insulin receptor substrate 1 (IRS-1) serine 307, and Akt phosphorylation, as well as cleaved caspase 3 levels in the mouse retina.

Results

We found that loss of TLR4 led to increased insulin receptor and Akt phosphorylation, as well as decreased IRS-1^Ser307 levels. In support of these results, TLR4 overexpression decreased insulin signaling and the cleavage of caspase 3.

Conclusions

Therefore, these results suggest that TLR4 plays a key role in insulin signaling in the retina. Reduction of TLR4 levels may be protective to the retina.

     

TLR4 mutation reduces microglial activation,increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease

Background

Amyloid plaques, a pathological hallmark of Alzheimer's disease (AD), are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs), a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start.

Methods

Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze.

Results

While no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg) was less than that in a TLR4 wild-type AD model (TLR4W Tg). At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes.

Conclusion

This is the first demonstration of TLR4-dependent activation of microglia at the early stage of β-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aβ deposition and that, as Aβ deposits start, microglia are activated via TLR4 signaling to reduce Aβ deposits and preserve cognitive functions from Aβ-mediated neurotoxicity.

     

Mast cells participate in allograft rejection: can IL-37 play an inhibitory role?

Objective

The aim of this study was to evaluate the role of mast cells (MCs) in allograft rejection, eventually inhibited by IL-37. Immune cells including MCs participate in allograft rejection by generating IL-1, IL-33, TNF and other cytokines.

Methods

We evaluated allograft rejection on the experience of our experimental data and using the relevant literature.

Results

MCs are involved in initiation and regulation of innate and adaptive immune responses-pathways. MCs are important pro-inflammatory cells which express high-affinity receptor FceRI and can be activated by IgE and some pro-inflammatory cytokines, such as IL-1 and IL-33. The cross-linkage of high affinity IgE receptor on MCs by antigen ligation has a crucial role in allergy, asthma, anaphylaxis, cancer and allograft rejection. MCs mediate immunity in organ transplant, leading to the activation of allospecific T cells implicated in the rejection and generate pro-inflammatory cytokines/chemokines. IL-1 pro-inflammatory cytokine family members released by MCs mediate allograft rejection and inflammation. IL-37 is also an IL-1 family member generated by macrophage cell line in small amounts, which binds to IL-18Rα and produces an anti-inflammatory effect. IL-37 provokes the inhibition of TLR signaling, TLR-induced mTOR and (MyD88)-mediated responses, suppressing pro-inflammatory IL-1 family members and increasing IL-10.

Conclusion

IL-37 inhibition offers the opportunity to immunologically modulate MCs, by suppressing their production of IL-1 family members and reducing the risk of allograft rejection, resulting as a potential good therapeutic new cytokine. Here, we report the relationship between inflammatory MCs, allograft rejection and pro-inflammatory and anti-inflammatory IL-37.

     

Inhibitory effects and related molecular mechanisms of total flavonoids in <Emphasis Type="Italic">Mosla chinensis</Emphasis> Maxim against H1N1 influenza virus

Objective

The Shixiangru (Mosla chinensis Maxim) total flavonoids (STF) mainly contain luteolin and apigenin. The study aims to examine the inhibitory effects of STF on anti-H1N1 influenza virus and its related molecular mechanisms in pneumonia mice.

Methods

The viral pneumonia mice were treated with Ribavirin or various doses of STF. We observed histological changes of lung by immunohistochemistry and measured lung index to value anti-influenza virus effects of STF. The concentrations of inflammatory cytokines and anti-oxidant factors were detected by ELISA. RT-PCR and western blot assays were used to determine the expression level of TLR pathway’s key genes and proteins in lung tissues.

Results

We found that the pathological changes of lung in the viral pneumonia mice obviously alleviated by STF treatments and the STF (288 or 576 mg/kg) could significantly decrease lung indices. Moreover, the up-regulation (IL-6, TNF-α, IFN-γ, and NO) and down-regulation (IL-2, SOD and GSH) of inflammatory cytokines and anti-oxidant factors were associated with higher clearance of virus and reduction of inflammatory lung tissue damage. Meanwhile, the expression levels of TLR3, TLR7, MyD88, TRAF3 and NF-κB p65 of the TLR pathway were reduced by STF treatment.

Conclusions

This study suggested that STF may be a promising candidate for treating H1N1 influenza and subsequent viral pneumonia.

     

Reduced monocyte adhesion to aortae of diabetic plasminogen activator inhibitor-1 knockout mice

Objective and design

To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions.

Methods and subjects

Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC).

Treatments

Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL).

Results

The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC.

Conclusion

The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.

     

Montelukast improves the changes of cytoskeletal and adaptor proteins of human podocytes by interleukin-13

Objective and design

Interleukin-13 (IL-13) has recently been reported to be a potential cytokine in the pathogenesis of minimal-change nephrotic syndrome (MCNS). However, the mechanistic insights associated with podocyte dysfunction mediated by IL-13-induced changes in various slit diaphragm (SD) and cytoskeletal molecules have not yet been shown in cultured human podocytes in vitro.

Materials

Human conditionally immortalized podocytes were used.

Treatment

Podocytes were incubated with various concentrations of IL-13 during the indicated time periods (6, 12, and 24 h) and montelukast was administered with the dose of 0.1 μg.

Results

Treatment of IL-13 resulted in a progressive decrease in distinct processes or projections of the human podocytes and high dose of IL-13 increased podocyte permeability in vitro at 6 h. IL-13 had a substantial impact on the redistribution and rearrangement of zonula occludens (ZO)-1, synaptopodin, α-actinin, CD2-associated protein (CD2AP) in podocytes and disrupted the cytoskeletal connections in a concentration-dependent manner on confocal microscopy. IL-13 also down-modulated ZO-1, synaptopodin, α-actinin, CD2AP, and p130Cas at protein levels and upregulated β-catenin and B7-1 in podocytes. Furthermore, we demonstrated that down-modulated changes in various SD and cytoskeletal structures of human podocytes induced by IL-13 was significantly restored after treatment with montelukast with upregulation of B7-1.

Conclusion

Our results suggest that targeting IL-13 may be one of the important cytokines in the pathogenesis of MCNS and targeting IL-13 could be one of the potential therapeutic strategies in MCNS.

     

Quetiapine ameliorates collagen-induced arthritis in mice via the suppression of the AKT and ERK signaling pathways

Objective and design

To investigate the amelioration effects of quetiapine on rheumatoid arthritis with RAW 264.7 macrophage and collagen-induced arthritis (CIA) DBA/1J mouse model.

Subjects

RAW 264.7 macrophage and DBA/1J mice.

Treatment

Lipopolysaccharide and collagen.

Methods

RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS) followed by quetiapine treatments were investigated. Activations of CD80 and CD86 were analyzed by flow cytometry. Pro-inflammatory cytokines such as IL-6, TNF-α and IL-1β were analyzed by ELISA. Proteins involved in signaling pathways related to the formation of rheumatoid arthritis were assayed by Western blotting. Therapeutic efficacy of quetiapine in CIA mouse model was also assayed. ¹⁸F-FDG/micro-PET was used to monitor the inflammation status in the joints, and the severity of bone erosion was evaluated with micro-CT and H&E staining.

Results

The inhibition of pro-inflammatory cytokines by quetiapine was found through the ERK and AKT phosphorylation and subsequent NF-κB and CREB signaling pathways. Pro-inflammatory cytokines such as IL-17, IL-6 and IL-1β were decreased, while immunosuppressive factors such as TGF-β and IL-10 were increased in CIA mice treated with quetiapine. Notably, no uptake of ¹⁸F-FDG and bone erosion was found with micro-PET images on days 32 and 43 in the quetiapine-treated and normal control groups. However, significant uptake of ¹⁸F-FDG could be observed in the CIA group during the same time course. Similar results were further verified with ex vivo autoradiography.

Conclusion

Taken together, these results suggest that quetiapine is a potential anti-inflammatory drug, and may be used as an adjuvant for the treatment of rheumatoid arthritis.

     

Involvement of Fas/FasL pathway in the murine model of atopic dermatitis

Objective and design

The aim of this study was to elucidate the role of apoptosis mediated through Fas/FasL pathway using the mouse model of atopic dermatitis (AD).

Materials and treatment

AD was induced by epicutaneous application of ovalbumin (OVA) in wild-type C57BL/6, B6. MRL-Faslpr/J (Fas?) and B6Smn.C3-Faslgld/J (FasL?) mouse strains.

Methods

Skin samples were subjected to staining for Fas/FasL expression, M30 epitope and assessment of inflammatory response via immunohistochemical staining. Cytokine and chemokine production was assessed by real-time PCR.

Results

In comparison to wild-type mice, OVA sensitization of Fas- and FasL-deficient mice led to increased epidermal and dermal thickness, collagen deposition and local inflammation consisting of macrophages, neutrophils and CD4+?T cells. Fas- and FasL-deficient mice showed increased total counts of regulatory T cells (Tregs) and IgE levels in blood as well as increased expression of IL-1β, IL-4, IL-5, IL-13 and TGF-1β mRNA in comparison to wild-type mice. On the other hand, expression of CXCL9 and CXCL10, IL-17 mRNAs in the skin samples in Fas- and FasL-deficient mice was decreased.

Conclusions

Our results show that lack of the Fas-induced apoptosis leads to exacerbation of AD characteristics such as Th2 inflammation and dermal thickening. Therefore, Fas receptor can play an important role in AD pathogenesis by controlling development of the local inflammation.

     

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation

Background

Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches.

Methods

Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects.

Results

AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS.

Conclusions

Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

     

Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients

Objective

To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients.

Methods

Spontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation.

Results

The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function.

Conclusions

These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

     

S100B promotes microglia M1 polarization and migration to aggravate cerebral ischemia

Aim and objective

S100B has been found abundantly expressed in microglia during cerebral ischemia. However, S100B effects on phenotype changes and migration of microglia are unclear.

Methods

Real-time PCR of S100B, M1 and M2 markers were tested to characterize phenotypic changes in microglia in mice middle cerebral artery occlusion (MCAO) model. Migration assay and additional mechanism studies were performed to elucidate the role of NF-κB in S100B-mediated microglia M1/M2 phenotype change and migration. Finally, S100B treatment on MCAO models was performed to show the in vivo evidence.

Results

S100B was identified as an induced gene with its pattern in accordance with M1 markers in mice MCAO model. That S100B was promoted by M1 stimuli whereas inhibited by M2 stimuli further confirmed S100B a M1 marker. Moreover, S100B promotes microglia M1 polarization with enhanced migration ability and inhibits M2 polarization. Additionally, NF-κB is essential in S100B control in microglia M1/M2 polarization and migration. Furthermore, S100B aggravated cerebral ischemia in murine MCAO model and exacerbated the microglia M1 polarization and migration.

Conclusions

Our findings demonstrate that S100B promotes microglia M1 polarization to aggravate cerebral ischemia, and provide a better understanding on the therapeutic effects of S100B and/or its antagonist/neutralization antibody in stroke.

     

The atypical RhoGTPase RhoE/Rnd3 is a key molecule to acquire a neuroprotective phenotype in microglia

Background

Over-activated microglia play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration. Reversion of over-activated to neuroprotective microglia phenotype could regenerate a healthy CNS-supporting microglia environment. Our aim was to identify a dataset of intracellular molecules in primary microglia that play a role in the transition of microglia to a ramified, neuroprotective phenotype.

Methods

We exploited the anti-inflammatory and neuroprotective properties of conditioned medium of adipose-derived mesenchymal stem cells (CM) as a tool to generate the neuroprotective phenotype of microglia in vitro, and we set up a microscopy-based siRNA screen to identify its hits by cell morphology.

Results

We initially assayed an array of 157 siRNAs against genes that codify proteins and factors of cytoskeleton and activation/inflammatory pathways in microglia. From them, 45 siRNAs significantly inhibited the CM-induced transition from a neurotoxic to a neuroprotective phenotype of microglia, and 50 siRNAs had the opposite effect. As a proof-of-concept, ten of these targets were validated with individual siRNAs and by downregulation of protein expression. This validation step resulted essential, because three of the potential targets were false positives. The seven validated targets were assayed in a functional screen that revealed that the atypical RhoGTPase RhoE/Rnd3 is necessary for BDNF expression and plays an essential role in controlling microglial migration.

Conclusions

Besides the identification of RhoE/Rnd3 as a novel inducer of a potential neuroprotective phenotype in microglia, we propose a list of potential targets to be further confirmed with selective activators or inhibitors.

     

Fluoxetine attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage: a possible role for the regulation of TLR4/MyD88/NF-κB signaling pathway

Background

Neuroinflammation is closely associated with functional outcome in subarachnoid hemorrhage (SAH) patients. Our recent study demonstrated that fluoxetine inhibited NLRP3 inflammasome activation and attenuated necrotic cell death in early brain injury after SAH, while the effects and potential mechanisms of fluoxetine on neuroinflammation after SAH have not been well-studied yet.

Methods

One hundred and fifty-three male SD rats were subjected to the endovascular perforation model of SAH. Fluoxetine (10?mg/kg) was administered intravenously at 6?h after SAH induction. TAK-242 (1.5?mg/kg), an exogenous TLR4 antagonist, was injected intraperitoneally 1?h after SAH. SAH grade, neurological scores, brain water content, Evans blue extravasation, immunofluorescence/TUNEL staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were performed.

Results

Fluoxetine administration attenuated BBB disruption, brain edema, and improved neurological function after SAH. In addition, fluoxetine alleviated the number of Iba-1-positive microglia/macrophages, neutrophil infiltration, and cell death. Moreover, fluoxetine reduced the levels of pro-inflammatory cytokines, downregulated the expression of TLR4 and MyD88, and promoted the nuclear translocation of NF-κB p65, which were also found in rats with TAK-242 administration. Combined administration of fluoxetine and TAK-242 did not enhance the neuroprotective effects of fluoxetine.

Conclusion

Fluoxetine attenuated neuroinflammation and improved neurological function in SAH rats. The potential mechanisms involved, at least in part, TLR4/MyD88/NF-κB signaling pathway.