The Experimental Autoimmune Encephalomyelitis Disease Course Is Modulated by Nicotine and Other Cigarette Smoke Components

<div><p>Epidemiological studies have reported that cigarette smoking increases the risk of developing multiple sclerosis (MS) and accelerates its progression. However, the molecular mechanisms underlying these effects remain unsettled. We have investigated here the effects of the nicotine and the non-nicotine components in cigarette smoke on MS using the experimental autoimmune encephalomyelitis (EAE) model, and have explored their underlying mechanism of action. Our results show that nicotine ameliorates the severity of EAE, as shown by reduced demyelination, increased body weight, and attenuated microglial activation. Nicotine administration after the development of EAE symptoms prevented further disease exacerbation, suggesting that it might be useful as an EAE/MS therapeutic. In contrast, the remaining components of cigarette smoke, delivered as cigarette smoke condensate (CSC), accelerated and increased adverse clinical symptoms during the early stages of EAE, and we identify a particular cigarette smoke compound, acrolein, as one of the potential mediators. We also show that the mechanisms underlying the opposing effects of nicotine and CSC on EAE are likely due to distinct effects on microglial viability, activation, and function.</p></div

Clondronate ablates microglia in organotypic hippocampal brain slices.

<p>Hippocampal slices were obtained from MacGreen mice and were treated with clodronate (5 µg/ml) or MIF (100 µg/ml) for 6 or 14 days and examined using confocal microscopy. <b>A.</b> Microglia could not be detected in clodronate-treated slices at DIV14, whereas microglia in MIF-treated slices appear similar in morphology to microglia in control slices. Scale bar, 20 µm. <b>B.</b> Western blot analysis of NeuN, Iba-1, and GFAP. Alpha-tubulin was used as a loading control.</p

Increased demyelination in CSC-treated mice at day 14.

<p>Frozen sections of spinal cords were isolated from vehicle (saline/DMSO) and CSC-treated mice at day 14 and day 28 of EAE. Eriochrome cyanine <b>(A)</b> or fluoromyelin <b>(B)</b> was used to visualize demyelination. Areas of demyelination are indicated by diminished fluorescence; boxed regions are shown at higher magnification. <b>(C)</b> Demyelinated areas from <b>(B)</b> were measured using ImageJ and calculated based on equation listed in Methods (n = 4, *p<0.05). The levels of demyelination at day 14 in saline- or nicotine-infused spinal cords are also shown for comparison.</p

Microglia decrease the levels of synaptic adhesion molecules.

<p>Hippocampal neurons at 19 DIV were co-cultured with microglia for 2 days. <b>A.</b> Western blot analysis of the levels of N-cadherin, pan-γ-protocadherin, and SynCAM-1 in neurons in the absence or presence of microglia. α-tubulin was used as a loading control. <b>B.</b> Quantification was performed using the ImageJ software and normalized against α-tubulin (n = 4). *P<0.05 compared to neurons alone.</p

Microglia Actively Regulate the Number of Functional Synapses

<div><p>Microglia are the immunocompetent cells of the central nervous system. In the physiological setting, their highly motile processes continually survey the local brain parenchyma and transiently contact synaptic elements. Although recent work has shown that the interaction of microglia with synapses contributes to synaptic remodeling during development, the role of microglia in synaptic physiology is just starting to get explored. To assess this question, we employed an electrophysiological approach using two methods to manipulate microglia in culture: organotypic hippocampal brain slices in which microglia were depleted using clodronate liposomes, and cultured hippocampal neurons to which microglia were added. We show here that the frequency of excitatory postsynaptic current increases in microglia-depleted brain slices, consistent with a higher synaptic density, and that this enhancement ensures from the loss of microglia since it is reversed when the microglia are replenished. Conversely, the addition of microglia to neuronal cultures decreases synaptic activity and reduces the density of synapses, spine numbers, surface expression of AMPA receptor (GluA1), and levels of synaptic adhesion molecules. Taken together, our findings demonstrate that non-activated microglia acutely modulate synaptic activity by regulating the number of functional synapses in the central nervous system.</p> </div

TNF-α levels are below detection levels in control or clodronate- or MIF-treated organotypic slices or in co-cultures of neurons and microglia.

<p>Media from control or clodronate- or MIF-treated organotypic slices at DIV6 and 14, and hippocampal neuronal cultures (1°neurons) in the absence (Neu) or presence (Neu+mic) of microglia for 2 days were collected. TNF-α levels were measured by ELISA. TNF-α levels in media collected from primary microglia exposed for 12 h to LPS (100 ng/ml) were used as a positive control.</p

Non-nicotine components of cigarette smoke and nicotine have opposite effects on microglial state during early stages of EAE.

<p>(<b>A</b>) Resting Iba1+ (M0), pro-inflammatory (M1) and anti-inflammatory (M2) microglia were identified by labeling with anti-iNOS/Iba1 and anti-Arg1/Iba1, respectively. iNOS^+/Iba1⁺ (<b>B</b>) and Arg1^+/Iba1⁺ (<b>C</b>) cells were counted and compared between vehicle (pooled DMSO/saline samples) (n = 4) and CSC (n = 3)/nicotine-treated (n = 3) samples. Percentages of each population after treatment are indicated in (<b>D</b>). Bar = 50 µm (**p<0.01).</p

Effects of nicotine and non-nicotine components of cigarette smoke on EAE severity.

<p>EAE was induced by injection of MOG_35–55 in CFA and pertussis toxin. <b>(A)</b> CSC was infused at 10 or 20 mg/ml into EAE mice starting at day 0 of EAE for 28 days, with DMSO as vehicle. <b>(B)</b> 20 mg/ml CSC was infused into the mice from 14 days prior to EAE induction through day 28, with 50% DMSO as vehicle. <b>(C)</b> Nicotine (200 mg/ml) was infused into EAE mice starting at Day 0 of EAE for 28 days, with saline as vehicle. Peak score <b>(D)</b> and cumulative score <b>(E)</b> were compared between CSC (n = 3)/nicotine (n = 6) and control mice (n = 8). (*** p<0.001; **p<0.01; *p<0.05).</p

Decreased demyelination in nicotine-treated mice during EAE.

<p><b>(A)</b> Frozen sections of spinal cords were isolated from vehicle (saline) and nicotine-treated mice at different time points during EAE. Eriochrome cyanine (EC) was used to visualize demyelination. Intact white matter (WM) is indicated by the blue staining and demyelination is revealed by diminished color. Areas of demyelination are indicated by asterisks; boxed regions are shown at higher magnification. <b>(B)</b> Demyelinated areas were measured using ImageJ and calculated based on equation listed in Methods. (n = 3–4, *p<0.05).</p

Therapeutic administration of nicotine attenuates EAE symptoms.

<p><b>(A)</b> 14-day pumps filled with 200 mg/ml nicotine were placed in the backs of EAE mice at day 14 (***<0.001). Peak score <b>(B)</b> as well as cumulative score <b>(C)</b> from day 14 to day 28 were recorded and compared between the vehicle (n = 8) and nicotine-treated (n = 3) groups. <b>(D)</b> Weekly weights were plotted as a percentage of day 0 weights. (***p<0.001; *p<0.05)</p