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The effects of repetitive magnetic stimulation (rMS) have predominantly been studied in excitable cells, with limited research in non-excitable cells. This study aimed to investigate the impact of rMS on macrophages, which are crucial cells in the innate immune defense. THP-1-derived macrophages subjected to a 5 min session of 10 Hz rMS exhibited increased Nrf2 activation and decreased Keap1 expression. We found that activation of the Nrf2 signaling pathway relied on rMS-induced phosphorylation of p62. Notably, rMS reduced the intracellular survival of Staphylococcus aureus in macrophages. Silencing Nrf2 using siRNA in THP-1-derived macrophages or utilizing Nrf2 knockout in alveolar macrophages abolished this effect. Additionally, rMS attenuated the expression of IL-1β and TNF-α inflammatory genes by S. aureus and inhibited p38 MAPK activation. These findings highlight the capacity of rMS to activate the non-canonical Nrf2 pathway, modulate macrophage function, and enhance the host’s defense against bacterial infection.

Abstract Cervical spinal cord injury (SCI) results in permanent life-altering motor and respiratory deficits. Other than mechanical ventilation for respiratory insufficiency secondary to cervical SCI, effective treatments are lacking and the development of animal models to explore new therapeutic strategies are needed. The aim of this work was to demonstrate the feasibility of using a mouse model of partial cervical spinal hemisection at the second cervical metameric segment (C2) to investigate the impact of 6 weeks training on forced exercise wheel system on locomotor/respiratory plasticity muscles. To measure run capacity locomotor and respiratory functions, incremental exercise tests and diaphragmatic electromyography were done. In addition, muscle fiber type composition and capillary distribution were assessed at 51 days following chronic C2 injury in diaphragm, extensor digitorum communis (EDC), tibialis anterior (TA) and soleus (SOL) muscles. Six-week exercise training increased the running capacity of trained SCI mice. Fiber type composition in EDC, TA and SOL muscles was not modified by our protocol of exercise. The vascularization was increased in all muscle limbs in SCI trained group. No increase in diaphragmatic electromyography amplitude of the diaphragm muscle on the side of SCI was observed, while the contraction duration was significantly decreased in sedentary group compared to trained group. Cross-sectional area of type IIa myofiber in the contralateral diaphragm side of SCI was smaller in trained group. Fiber type distribution between contralateral and ipsilateral diaphragm in SCI sedentary group was affected, while no difference was observed in trained group. In addition, the vascularization of the diaphragm side contralateral to SCI was increased in trained group. All these results suggest an increase in fatigue resistance and a contribution to the running capacity in SCI trained group. Our exercise protocol could be a promising non-invasive strategy to sustain locomotor and respiratory muscle plasticity following SCI.

High spinal cord injuries (SCI) induce the deafferentation of phrenic motoneurons, leading to permanent diaphragm paralysis. This involves secondary injury associated with pathologic and inflammatory processes at the site of injury, and at the level of phrenic motoneurons. In the present study, we evaluated the antioxidant response in phrenic motoneurons involving the AMPK-Nrf2 signaling pathway following C2 spinal cord lateral hemi-section in rats. We showed that there is an abrupt reduction in the expression of phosphorylated AMPK and Nrf2 at one hour post-injury in phrenic motoneurons. A rebound is then observed at one day post-injury, reflecting a return to homeostasis condition. In the total spinal cord around phrenic motoneurons, the increase in phosphorylated AMPK and Nrf2 occurred at three days post-injury, showing the differential antioxidant response between phrenic motoneurons and other cell types. Taken together, our results display the implication of the AMPK-Nrf2 signaling pathway in phrenic motoneurons’ response to oxidative stress following high SCI. Harnessing this AMPK-Nrf2 signaling pathway could improve the antioxidant response and help in spinal rewiring to these deafferented phrenic motoneurons to improve diaphragm activity in patients suffering high SCI.

High cervical spinal cord injuries lead to permanent respiratory deficits. One preclinical model of respiratory insufficiency in adult rats is the C2 partial injury which causes unilateral diaphragm paralysis. This model allows the investigation of a particular population of respiratory bulbospinal axons which cross the midline at C3-C6 spinal segment, namely the crossed phrenic pathway. Transcranial magnetic stimulation (TMS) is a non-invasive technique that can be used to study supraspinal descending respiratory pathways in the rat. Interestingly, a lateral C2 injury does not affect the amplitude and latency of the largest motor-evoked potential recorded from the diaphragm (MEPdia) ipsilateral to the injury in response to a single TMS pulse, compared to a sham animal. Although the rhythmic respiratory activity on the contralateral diaphragm is preserved at 7 days post-injury, no diaphragm activity can be recorded on the injured side. However, a profound reorganization of the MEPdia evoked by TMS can be observed. The MEPdia is reduced on the non-injured rather than the injured side. This suggests an increase in ipsilateral phrenic motoneurons excitability. Moreover, correlations between MEPdia amplitude and spontaneous contralateral diaphragmatic activity were observed. The larger diaphragm activity correlated with a larger MEPdia on the injured side, and a smaller MEPdia on the non-injured side. This suggests, for the first time, the occurrence of a functional neuroplasticity process involving changes in motoneuron excitability balance between the injured and non-injured sides at a short post-lesional delay.

Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their potential or proven anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-1-derived macrophages. We first compared the capacity of sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), and dimethyl fumarate (DMF) to induce the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. Next, we performed a comparative evaluation of the antioxidant and anti-inflammatory efficacies of the 4 selected compounds. THP-1-derived macrophages and LPS-stimulated macrophages were treated with each compound and expression levels of genes coding for inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified by RT-qPCR. Moreover, expression levels of genes coding for M1 (IL-23, CCR7, IL-1β, IL-6, and TNF-α) and M2 (PPARγ, MRC1, CCL22, and IL-10) markers were determined in classically-activated M1 macrophages treated with each compound. Finally, the effects of each compound on the intracellular bacterial survival of gram-negative E. coli and gram-positive S. aureus in THP-1-derived macrophages and PBMC-derived macrophages were examined. Our data confirmed the anti-inflammatory and antioxidant effects of SFN, WG, and DMF on LPS-stimulated THP-1-derived macrophages. In addition, SFN or WG treatment of classically-activated THP-1-derived macrophages reduced expression levels of M1 marker genes, while SFN or DMF treatment upregulated the M2 marker gene MRC1. This decrease in expression of M1 marker genes may be correlated with the decrease in intracellular S. aureus load in SFN- or DMF-treated macrophages. Interestingly, an increase in intracellular survival of E. coli in SFN-treated THP-1-derived macrophages that was not observed in PBMC-derived macrophages. Conversely, OTZ exhibited pro-oxidant and proinflammatory properties, and affected intracellular survival of E. coli in THP-1-derived macrophages. Altogether, we provide new potential therapeutic alternatives in treating inflammation and bacterial infection.