Atmospheric particulate matter aggravates CNS demyelination via TLR-4/NF-κB-mediated microglia pathogenic activities

Atmospheric Particulate Matter (PM) is one of the leading environmental risk factors for the global burden of disease. Increasing epidemiological studies demonstrated that PM plays a significant role in CNS demyelinating disorders; however, there is no direct testimony of this, and yet the molecular mechanism by which the occurrence remains unclear. Using multiple in vivo and in vitro strategies, in the present study we demonstrate that PM exposure aggravates neuroinflammation, myelin injury, and dysfunction of movement coordination ability via boosting microglial pro-inflammatory activities, in both the pathological demyelination and physiological myelinogenesis animal models. Indeed, pharmacological disturbance combined with RNA-seq and ChIP-seq suggests that TLR-4/NF-κB signaling mediated a core network of genes that control PM-triggered microglia pathogenicity. In summary, our study defines a novel atmospheric environmental mechanism that mediates PM-aggravated microglia pathogenic activities, and establishes a systematic approach for the investigation of the effects of environmental exposure in neurologic disorders. Significance An atmospheric trigger, the respirable particulate matter boost microglia pathogenic activities in the context of CNS demyelination by activating TLR-4/NF-κB signaling axis in animal models of immune- and toxicity-induced demyelination, as well as myelinogenesis during postnatal development.

Animal treatment 113 Considering the actual population exposure dose and the previous study (Ku et al. 2017 and 5, death. EAE mice were randomly enrolled in the following treatment groups: 1) 135 Sham-treated PBS control group: EAE mice were exposed intranasally to 20 μL PBS; 2) 136 PM-treated group: EAE mice were exposed to 10.0 mg/mL PM in a total of 20 μL PBS    Behavioral experiments 162 In order to evaluate the motor balance and motor coordination of mice, beam walking test, 163 rota-rod test, and wire hang test were done at the postnatal 19 th , 20 th and 21 th day, 164 respectively. Beaming walking test and wire hang test have been done between 8a.m. to 165 12a.m, and rota-rod test has been completed between 2p.m to 6p.m. After each trail, all 166 devices were wiped clean with 75% alcohol to prevent interference with the next trail. (cut-off time 60 s). In the results, the mean score of the two trials is given.

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In the rotarod test (Scoles et al. 2017), mouse was put to the rotarod with a speed of 174 5 rpm for 5min one day in advance to adapt to the rotarod. In the formal trial, mice were 175 placed on the rotarod which has an initial speed of 5 rpm and accelerates at an increase of 1 rpm per second. The latency that the mouse stuck before they fell was recorded. Mice 177 received two trials which each trial interval 60s, and the mean score of the two trials is 178 given in the final results.          Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts.

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In this step, clean data (clean reads) were obtained by removing reads containing adapter, 328 reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 329 and GC content the clean data were calculated. All the downstream analyses were based 330 on the clean data with high quality. STAR is used to align clean reads to reference genome.  Fisher's exact test was applied to identify the significant GO categories and FDR was 348 used to correct the p-values. Pathway analysis was used to find out the significant 349 pathway of the differential genes according to KEGG database. We turn to the Fisher's 350 exact test to select the significant pathway, and the threshold of significance was defined 351 by P-value and FDR. 354 Approximately 1.2 million microglia were exposed to the indicated treatments    Figure 1E). The total number of MNCs per mouse in the 419 PM-treated group was 13.9 ± 1.1 × 10 6 , which is ~2.2-fold of that PBS control (6.24 ± 420 0.8 × 10 6 , p< 0.001, Figure 1E). We then determined the effect of PM on myelin loss 421 using anti-MBP (myelin marker) staining. As shown in Figure 1F, a significant degree   Figure 2D).

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PM aspiration not only significantly reduced the number of myelination axon, but also 471 increased G-ratio of the remyelinated axons, indicating a poor recovery from 472 demyelination ( Figure 2E). These findings suggest that PM aspiration exacerbates 473 demyelination and prevents remyelination in toxin-induced demyelination model. walking, and tight rope test. In the rotating rod test at accelerating speed, PM-treated 504 mice exhibited a significant shorter latency to fall off the rod than that of PBS-treated 505 mice (decreased -32.6% in the time of staying on the rotating rod, Figure 3G, p<0.001). Disturbances of motor dysfunction in PM-treated mice were also observed 507 in the beam walking and tight rope test. Mice exposed to PM had significantly worse 508 motor performance, as the time taken by mice to traverse a narrow beam or cotton 509 rope increased remarkably compared to PBS-treated control mice ( Figure 3G, p<0.05).

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Although little is known about the mechanism of PM exacerbating myelin damage, 518 we have observed a significant increase of activated IBA1+ microglia in the 519 demyelination/myelinogenesis animal models ( Figure 1F and G, Figure 2C, Figure 3B Figure 5J). Together, these data suggest that NF-κB-associated 618 inflammation signaling mediated a core network of genes that control PM-triggered 619 microglia priming. understood. Therefore, to create targeted and effective therapies, mechanism of action 633 (e.g., specific signaling transduction and responsors) that mediates the exacerbating or 634 mitigating clinical symptoms of disease needs to be elaborated.

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In the present study, we aim to identify critical cellular and molecular targets that 636 alter demyelination disease after PM exposure, to identify the therapeutic strategies 637 that may be particularly applicable to patients who are exposed to high levels of PM.
We demonstrate that PM exposure exacerbates CNS myelin injury, based on three   Taken together, our present study confirms that PM inhalation leads to aggravate CNS 703 demyelination, and this action is associated with a previously unrecognized role for TLR4/NF-κB signaling-mediated microglia activation. The results suggest a novel 705 mechanism for PM-produced adverse effects on the nervous system and present a 706 potential intervention target for prevention. Importantly, given the specific nature of 707 PM, e.g., the biologic responses of this complex mixtures is further influenced by the 708 source and constituents, the route of exposure, the particulate matrix within which 709 they reside, the potential different mechanisms and bio-availability of these 710 components, and the genetic differences of the recipients, we believe that the 711 investigation of these differences is necessary to clarify the characteristics of PM 712 exposure and the potential to cause CNS disease. Only then will it be reasonable to 713 propose targeted remediation to stem the tide of demyelination disease that is growing 714 in populations facing air pollution.