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Sneaky Entry of Ifnγ Through Arsenic-Induced Leaky Blood–Brain Barrier Reduces CD200 Expression by Microglial Pro-Inflammatory Cytokine

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Sneaky Entry of Ifnγ Through Arsenic-Induced Leaky Blood–Brain Barrier Reduces CD200 Expression by Microglial Pro-Inflammatory Cytokine Molecular Neurobiology (2019) 56:1488–1499 https://doi.org/10.1007/s12035-018-1155-0 Sneaky Entry of IFNγ Through Arsenic-Induced Leaky Blood–Brain Barrier Reduces CD200 Expression by Microglial pro-Inflammatory Cytokine Vikas Singh1,2 & Shaivya Kushwaha1,2 & Ruchi Gera1,2 & Jamal Ahmad Ansari1,2 & Juhi Mishra3 & Jayant Dewangan4 & Satyakam Patnaik2,5 & Debabrata Ghosh1,2 Received: 13 January 2018 /Accepted: 25 May 2018 /Published online: 12 June 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Recent studies showed that neuronal surface protein CD200 plays a key role in the regulation of neuroinflammation. Previously, we showed that arsenic (0.38 mg/kg body weight) exposure induces microglial activation and consequently IL-6/TNF-α secre- tion. This result indicated the possibility of alteration in the expression of CD200. Therefore, the present study was focused on checking arsenic-induced alteration in CD200 expression and revealing the underlying mechanism. Male BALB/c mice were exposed to arsenic (vehicle, 0.038 and 0.38 mg/kg body weight) for 60 days, and the expression level of CD200 was found to be decreased which was rescued by minocycline (33 mg/kg body weight) co-administration. Higher CD68 staining, increased level of IL-6/TNF-α, as well as higher level of IFNγ, were observed in in vivo arsenic-exposed groups. Interestingly, in vitro arsenic exposure could not increase IL-6/TNF-α level in the culture supernatant, whereas, supplementation of IFNγ could mimic the in vivo results. However, arsenic could not induce IFNγ production from brain endothelial cells, microglia, and astrocytes, thereby suggesting the entry of IFNγ through the impaired blood–brain barrier. Evans blue fluorescence in the brain confirms altered blood–brain barrier permeability although no changes were observed in the expression level of tight junction proteins (claudin-5 and occludin). Finally, intracerebral injection of anti-IFNγ neutralizing antibody in arsenic-exposed brain reduced microglia activation (IL-6 and TNF-α and CD68 expression) and subsequently rescued CD200 level. Taken together, the study showed that arsenic-mediated compromised blood–brain barrier is a major driving force to induce microglial IL-6 and TNF-α production through serum IFNγ leading to CD200 downregulation. Keywords Microglia . Cytokine . Neuron . CD200 . IFNγ . Arsenic . Blood–brain barrier Introduction * Debabrata Ghosh debabrataghosh78@gmail.com; debabrata.ghosh@iitr.res.in Neuroinflammation is a tightly controlled phenomenon and kept in check by different inhibitory signals which otherwise 1 Immunotoxicology Laboratory, Food, Drug & Chemical Toxicology Group, and Nanotherapeutics & Nanomaterial Toxicology Group, can lead to different disease state in the central nervous sys- CSIR-Indian Institute of Toxicology Research (CSIR-IITR), tem. Contact-dependent cell-cell interaction between microg- Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, P.O. Box No. 80, lia and neurons through CD200R and CD200 is one of the Lucknow, Uttar Pradesh 226001, India major inhibitory signals [1]. Chronic neuroinflammatory sig- 2 Academy of Scientific and Innovative Research (AcSIR), CSIR-IITR nals downregulate CD200 expression and thereby sensitize campus, Lucknow 226001, India the brain towards neurodegenerative diseases. CD200 expres- 3 Developmental toxicology Laboratory, Systems Toxicology & sion was found to be decreased in the patients with pediatric Health Risk Assessment Group, CSIR-Indian Institute of Toxicology epilepsy, Parkinson’s, as well as Alzheimer’sdisease[2–4]. Research, Lucknow, Uttar Pradesh 226001, India Experimental CD200−/− mouse showed compromised blood– 4 Genotoxicity Lab, CSIR-Central Drug Research Institute, Uttar brain barrier (BBB) leading to IFNγ entry into the brain [5] Pradesh, Lucknow 226031, India and showed enhanced susceptibility to encephalomyelitis [6]. 5 Water Analysis Laboratory, Nanotherapeutics and Nanomaterial In contrary, CD200 fusion protein (CD200Fc) shown to re- Toxicology Group, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh 226001, India duce the neuroinflammation [7] as well as inhibit the Mol Neurobiol (2019) 56:1488–1499 1489 induction of experimental autoimmune uveoretinitis (EAU) Materials and Methods [3]. CD200 expression was observed to be increased both in vitro and in vivo in mouse neurons in the presence of anti- Reagents and Antibodies inflammatory cytokine IL-4 [8]. Endogenous factors like Aβ decrease CD200 expression Sodium arsenite (SA) (NaAsO2), Percoll, cell culture medium [9]. Apart from its role in neuroinflammation, promoters of (DMEM/F12), papain, sodium dodecyl sulphate (SDS), acryl- both the human and mouse CD200 gene have a binding site amide, bis-acrylamide, bromophenol blue, Tween 20, N- for tumor suppressor protein p53 thereby showing its involve- ethylmaleimide, o-phthalaldehyde, EDTA, protease inhibitor ment in apoptosis [10]. The transcription factor C/EBPβ reg- cocktail, 10× Western blocking buffer, methanol (LC grade), ulates the constitutive expression of CD200 by binding to its minocycline, Evans Blue, Cytosine arabinoside, and Poly L promoter [11]. Along with endogenous factors, various exog- Lysine were obtained from Sigma (St. Louis, MO, USA). enous factors like bacterial cell wall component, LPS, as well Fetal bovine serum (FBS) was purchased from Cell Clone as various environmental toxicants can alter the expression of Inc. PVDF membrane, Occludin antibody, claudin-5 antibody, CD200. Among various environmental chemicals, arsenic is and chemiluminescence substrates were obtained from globally infamous for its toxicity in every sphere of life [12, Merck-Millipore (USA). CD200 antibody was purchased 13]. Arsenic is known to cross blood–brain barrier (BBB) and from Santacruz biotechnology. CD68 antibody is obtained affect neuronal health in exposed animal [14]. The study in- from Abcam. IFNγ neutralizing antibody, N-2 supplement, dicates that arsenic may affect the integrity of BBB and alter B27 supplement, B5 supplement, Glutamax, neurobasal me- the permeability. Our group has reported that environmentally dium, and Alexa Fluor 594 anti-rabbit were obtained from relevant arsenic dose (0.38 ppm) induces pro-inflammatory Thermo Scientific. Recombinant IFNγ was purchased from cytokines like IL-6 and TNF-α in ex vivo microglia [15]. BD Biosciences. Tissue-freezing medium was obtained from Therefore, there is high chance that arsenic would affect the Leica. expression of CD200 in the brain. There are reports showing compromised cell-cell interaction result in the modest release of inflammatory cytokine from microglia causing cognitive Animals and Treatment impairment. On the other hand, lower IQ, decreased learning and memory response, as well as altered cognitive response Six to eight-week old male BALB/c mice (20–25 g) were were observed among individuals in arsenic-affected areas procured from the CSIR-Indian Institute of Toxicology [16]. Therefore, environmental stress-mediated dysfunction Research (CSIR-IITR) animal facility. All the protocols of neuron-microglial interaction impelled us to explore the for this study were approved by the Institutional Animal mechanism of arsenic-induced alteration in CD200 Ethics Committee of CSIR-IITR, Lucknow, India, and all expression. experiments have been carried out in accordance with the In the present study, we tried to find out the role of arsenic in guidelines laid down by the committee for control and altering the expression of CD200 in the brain. Expression of supervision of experiments on animals (CPCSEA), CD200 was checked in the brain sample of 60-day-exposed Ministry of Environment and Forests (Government of mice with or without minocycline treatment as well as in primary India), New Delhi, India. Mice were housed at 25 °C with cortical neurons following in vitro arsenic exposure. Microglial food and water supplied ad libitum. Three sets of animal activation status was checked by immunostaining the activation experiments were performed, and animals were divided marker CD68 and cytokine (IL-6, TNF-α,andIFNγ) secretion randomly by investigator who is blind about treatment by primary microglia isolated from experimental mice using groups; in the first set, animals were divided into three multiplex cytokine detection kit. Level of IFNγ was also mea- groups: control, 0.038 ppm, and 0.38 ppm arsenic treat- sured in ex vivo microglia from an adult mouse, neonatal mi- ment group, in the second set, animals were divided into croglia, astrocyte, and endothelial cell culture supernatant as well three groups: control, 0.38 ppm arsenic, and 0.38 ppm ar- as in serum. The expression level of blood–brain barrier proteins, senic + minocycline (33 mg/kg) [17],andinthethirdset, i.e., occludin and claudin-5 were checked by western blot anal- animals were divided into three groups: vehicle control ysis. The integrity of the blood–brain barrier was checked by (sham control), 0.38 ppm arsenic, and 0.38 ppm arsenic + Evans blue dye permeability assay. Finally, the involvement of anti-IFNγ neutralizing antibody. Sodium arsenite solution IFNγ in the arsenic-mediated regulation of CD200 expression was gavage fed daily in the arsenic treatment groups. In the wasconfirmedbyinjectinganti-IFNγ antibody into the brain by second set, minocycline was given for the last 2 weeks stereotaxy. Thus, in the present study, we unraveled that arsenic every alternate day till sacrifice, and in the third set, exposure compromised the BBB
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