BackgroundIn vivo studies have demonstrated the ability of multi-walled carbon nanotubes (MWCNT) to induce airway remodeling, a key feature of chronic respiratory diseases like asthma and chronic obstructive pulmonary disease. However, the mechanism leading to remodeling is poorly understood. Particularly, there is limited insight about the role of airway epithelial injury in these changes.ObjectivesWe investigated the mechanism of MWCNT-induced primary human bronchial epithelial (HBE) cell injury and its contribution in inducing a profibrotic response.MethodsPrimary HBE cells were exposed to thoroughly characterized MWCNTs (1.5-24 μg/mL equivalent to 0.37-6.0 μg/cm2) and MRC-5 human lung fibroblasts were exposed to 1:4 diluted conditioned medium from these cells. Flow cytometry, ELISA, immunostainings/immunoblots and PCR analyses were employed to study cellular mechanisms.ResultsMWCNT induced NLRP3 inflammasome dependent pyroptosis in HBE cells in a time- and dose-dependent manner. Cell death and cytokine production were significantly reduced by antioxidants, siRNA to NLRP3, a caspase-1 inhibitor (z-WEHD-FMK) or a cathepsin B inhibitor (CA-074Me). Conditioned medium from MWCNT-treated HBE cells induced significant increase in mRNA expression of pro-fibrotic markers (TIMP-1, Tenascin-C, Procollagen 1, and Osteopontin) in human lung fibroblasts, without a concomitant change in expression of TGF-beta. Induction of pro-fibrotic markers was significantly reduced when IL-1β, IL-18 and IL-8 neutralizing antibodies were added to the conditioned medium or when conditioned medium from NLRP3 siRNA transfected HBE cells was used.ConclusionsTaken together these results demonstrate induction of a NLRP3 inflammasome dependent but TGF-beta independent pro-fibrotic response after MWCNT exposure.

The predictive coding model suggests that voice motor control is regulated by a process in which the mismatch (error) between feedforward predictions and sensory feedback is detected and used to correct vocal motor behavior. In this study, we investigated how predictions about timing of pitch perturbations in voice auditory feedback would modulate ERP and behavioral responses during vocal production. We designed six counterbalanced blocks in which a +100 cents pitch-shift stimulus perturbed voice auditory feedback during vowel sound vocalizations. In three blocks, there was a fixed delay (500, 750 or 1000 ms) between voice and pitch-shift stimulus onset (predictable), whereas in the other three blocks, stimulus onset delay was randomized between 500, 750 and 1000 ms (unpredictable). We found that subjects produced compensatory (opposing) vocal responses that started at 80 ms after the onset of the unpredictable stimuli. However, for predictable stimuli, subjects initiated vocal responses at 20 ms before and followed the direction of pitch shifts in voice feedback. Analysis of ERPs showed that the amplitudes of the N1 and P2 components were significantly reduced in response to predictable compared with unpredictable stimuli. These findings indicate that predictions about temporal features of sensory feedback can modulate vocal motor behavior. In the context of the predictive coding model, temporally-predictable stimuli are learned and reinforced by the internal feedforward system, and as indexed by the ERP suppression, the sensory feedback contribution is reduced for their processing. These findings provide new insights into the neural mechanisms of vocal production and motor control.

The present study used event-related potential (ERP) recordings to investigate the neural mechanisms of sensorimotor adaptation in response to altered auditory feedback (AAF) during vocal production. 12 healthy speakers were tested under a vocal motor adaptation paradigm in which the fundamental frequency (F0) of their voice auditory feedback was pitch-shifted downward by one semi-tone (- 100 cents) during vowel vocalizations. Behavioral results revealed that subjects adapted to AAF by producing opposing (upward) responses to pitch-shift stimuli, and this adaptive behavior persisted after feedback alteration was removed (washout). We found that adaptation to AAF was accompanied by a significant increase in the amplitude of a parietal ERP activity elicited after the onset of vocalization. However, no such effect was observed for pre-motor ERPs elicited before vocalization onset. Moreover, we found that adaptive vocal responses were negatively correlated with ERPs over the parietal and positively correlated with those over the fronto-central areas after vocalization onset. These findings suggest that vocal motor adaptation is mediated by sensorimotor reprogramming of feedforward motor commands through incorporating auditory feedback, which is indexed by modulation of behavioral and ERP responses to AAF. We suggest that modulation of neural activities in the parietal cortex highlights its significance as a neural interface for sensorimotor integration and indicates its critical role in vocal motor adaptation. Our findings support the notion that the parietal mechanisms are involved in driving adaptive motor behavior to cope with unexpected changes in the sensory environment to accomplish communication goals during vocal production and motor control.

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