Abstract

   To increase the imaging resolution and detection capability, the field
   strength of static magnetic fields (SMFs) in magnetic resonance imaging
   (MRI) has significantly increased in the past few decades. However,
   research on the side effects of high magnetic field is still very
   inadequate and the effects of SMF above 1 T (Tesla) on B cells have
   never been reported. Here, we show that 33.0 T ultra‐high SMF exposure
   causes immunosuppression and disrupts B cell differentiation and
   signaling. 33.0 T SMF treatment resulted in disturbance of B cell
   peripheral differentiation and antibody secretion and reduced the
   expression of IgM on B cell membrane, and these might be intensity
   dependent. In addition, mice exposed to 33.0 T SMF showed inhibition on
   early activation of B cells, including B cell spreading, B cell
   receptor clustering and signalosome recruitment, and depression of both
   positive and negative molecules in the proximal BCR signaling, as well
   as impaired actin reorganization. Sequencing and gene enrichment
   analysis showed that SMF stimulation also affects splenic B cells'
   transcriptome and metabolic pathways. Therefore, in the clinical
   application of MRI, we should consider the influence of SMF on the
   immune system and choose the optimal intensity for treatment.
     __________________________________________________________________

   33.0 T SMF treatment resulted in disturbance of B cell peripheral
   differentiation and antibody secretion, but lower magnetic fields,
   including 28.7, 17.8, or the 11.2 T SMFs had much less or no such
   effects. 33.0 T SMF treatment induced downregulate BCR proximal
   signaling and impaired F‐actin remodeling related BCR clustering. SMF
   stimulation affected the transcriptome and metabolic pathways of B
   cells.

   graphic file with name MCO2-4-e379-g007.jpg

   .

1. INTRODUCTION

   The static magnetic field (SMF) is generated by steady currents or
   permanent magnets with constant magnetic field intensity and
   distribution. The intensity of magnetic fields varies in different
   situations, such as the earth's magnetic field is about 0.5 G (Gauss),
   which is 50 μT (Microtesla). In the last few decades, SMF has been
   widely applied in medicine, such as magnetic resonance imaging (MRI),
   which is one of the most common clinical diagnostic equipment due to
   its powerful imaging capability. Higher magnetic field intensity of MRI
   has been pursued to increase the resolution and sensitivity while
   reducing exposure time.[58] ^1 In most hospitals, MRI strength ranges
   from 1.5 to 3.0 T, which has been proven to be nonhazardous.[59] ^2 The
   strongest human MRI scanner of 10.5 T has been tested, and a 21.1 T
   small‐bore MRI has also been used on rodents.[60] ^3 , [61]^4 The
   biosafety of SMF is crucial to its application. Previous studies have
   shown that SMF has a certain influence on animal reproduction,[62] ^5 ,
   [63]^6 behavior,[64] ^7 , [65]^8 nerve,[66] ^9 and immune system.[67]
   ^10 And these effects are related to magnetic field intensity,
   direction, and gradient as well as sample exposure time.[68] ^11 In
   biological effect studies, SMFs can be classified as weak, moderate,
   high, and ultra‐high in accordance with their magnetic intensity, which
   are less than 1 mT, 1 mT to 1 T, 1−20 T, and above 20 T,
   respectively.[69] ^11 To date, the SMF biosafety studies have mainly
   focused on magnetic fields below 20 T, while studies above 20 T are
   very limited. Tian et al.[70] ^12 examined the blood biochemistry,
   complete blood counts, key organ weights, and histomorphology of mice
   exposed to 7.0–33.0 T and found most parameters in the normal range.
   However, more thorough studies are still needed to investigate the
   biological effects of ultra‐high magnetic fields. In this study, we
   aimed to investigate the effects of exposure to ultra‐high SMF,
   especially with intensity ≥30 T in healthy adult mice. Four different
   magnetic field intensity stimulations were selected, 33.0 and 28.7 T,
   belonging to the ultra‐high SMF, while 17.8 and 11.2 T as controls,
   belonging to the high SMF group.

   The immune system plays an important role in host defense as well as
   tissue homeostasis,[71] ^13 and SMF has been shown to affect the immune
   system. SMF has potential antineoplastic[72] ^14 and antiphlogistic
   influences,[73] ^15 and SMFs with different parameters have various
   effects on the weight of immune organs and the total count of white
   blood cells in mice.[74] ^12 , [75]^16 , [76]^17 As for innate immune
   cells, 0.6 T magnetic field[77] ^18 can promote M2 polarization,
   probably because magnetism regulates the cytoskeleton by affecting the
   cell membrane and calcium channel receptor.[78] ^10 , [79]^19 10 T SMF
   exposure can reduce pro‐inflammatory cytokines secreting, such as IL‐6,
   IL‐8, and TNF‐α, and increase the secretion of anti‐inflammatory
   factors IL‐10 to some extent.[80] ^20 In addition, a previous study
   reported that 0.4 T SMF increased NK cell cytotoxicity by activating
   multiple mitogen‐activated protein kinase pathways.[81] ^21 As for
   adaptive immune cells, magnetic exposure remarkably increased the
   apoptosis of phytohemagglutinin‐stimulated lymphocytes.[82] ^20 This
   might be due to the genotoxicity of SMF, its effects on microtubule
   assembly and energy metabolism disrupting cell division.[83] ^14 ,
   [84]^20 , [85]^22 The susceptibilities of naïve and memory CD4^+ or
   CD8^+ T cells to SMF exposure were different.[86] ^20 0.5 mT SMF
   exposure decreased IFN‐γ secretion, cell proliferation, and
   intracellular free calcium concentration in memory CD4^+ T cells.[87]
   ^23 0.3 and 0.6 T SMFs enhanced the production of Granzyme B, IFN‐γ and
   TNF‐α in CD8^+ T cells while promoting mitochondrial respiration to
   increase antitumor function.[88] ^24 Although studies have recognized
   the importance of the influence of SMF exposure on the immune system,
   research has yet to systematically explore its effect on B lymphocytes.

   B lymphocytes are essential for humoral immunity and play a crucial
   role in antibody secretion, antigen presentation, and immune
   regulation. The activation of B cell receptor (BCR) is essential for B
   cell function. The binding of specific antigens to the BCRs induces
   their aggregation by cross‐linking BCRs, then the conformation of BCR
   changes to expose the activation site, which triggers B cell activation
   and initiates an array of intracellular signal cascades.[89] ^25 In the
   proximal signaling, the phosphorylated immunoreceptor tyrosine‐based
   activation motifs in Igα/β recruit and activate spleen tyrosine kinase
   and further recruit Bruton's tyrosine kinase (Btk), phospholipase C‐γ,
   and other downstream molecules with the help of lipid
   phosphatidylinositol 3,4,5‐trisphosphate.[90] ^26 In addition, Vav
   activates the Wiskott‐Aldrich Syndrome protein (WASp, an actin
   nucleation‐promoting factor) to induce actin aggregation, and the
   cytoskeleton reorganization regulates BCR aggregation and provides a
   feedback regulation on the BCR signaling.[91] ^27 The normal function
   of B cells is crucial to the immune system. It remains unclear whether
   SMF exposure affects BCR signaling transduction and actin
   reorganization.

   In this study, we explored the effects of SMF on B cell differentiation
   by exposing mice to SMFs with four different intensities. Through flow
   cytometry assay, we found that ultra‐high SMF altered the
   differentiation of peripheral B cells and decreased the expression of
   IgM on B cell surface compared with high SMF, while no influence on the
   proliferation and apoptosis of B cells was observed. Meanwhile, we
   noticed that the proximal BCR positive and negative molecules,
   including CD19, Btk, and SHIP, were decreased after antigen stimulation
   in the ultra‐high SMF exposure group. With total internal reflection
   fluorescence microscopy (TIRFm), we found decreased BCR aggregation,
   impaired B cell expansion, and abnormal actin reorganization after
   antigen stimulation in the mice of ultra‐high SMF exposure group.
   Therefore, our study suggests that ultra‐high SMFs could disrupt B cell
   peripheral differentiation as well as negatively regulate BCR signaling
   and thereby cause immunosuppression.

2. RESULTS

2.1. Ultra‐high SMF interferes with the differentiation of peripheral B cells
and inhibits antibody secretion

   We first examined the differentiation of peripheral B cells through
   flow cytometry under 33.0, 28.7, 17.8, and 11.2 T magnetic field
   exposure and found that compared with the sham group, the percentage of
   marginal zone (MZ) B cells had no significant changes (Figures [92]1A
   and [93]D). However, we found a slight effect on the development of
   follicular (FO) and transitional type‐1 (T1) B cells at 33.0 T, which
   was not detected at other intensities (Figures [94]1B, E, and [95]F).
   The percentage of T2 subgroups was significantly lower in both 33.0 T
   and 28.7 T ultra‐high SMF groups than their paired sham groups, while
   no differences were found at the other two high magnetic fields
   (Figures [96]1B and [97]G). In addition, we also found a reduced
   percentage of the germinal center (GC) subset in the 17.8 T group
   (Figure [98]1C and [99]H). Interestingly, we also found that 33.0 and
   28.7 T magnetic fields exposure could reduce the IgM expression in
   splenic B cells, but 17.8 and 11.2 T SMF exposure showed no obvious
   effect (Figure [100]1I). Therefore, we examined the mean fluorescence
   intensity (MFI) of IgM in subsets of peripheral B cells, which
   indicated that 33.0 T SMF exposure reduced IgM expression in MZ and T2
   B cells and 28.7 T SMF exposure reduced that in FO, MZ, and T1 B cells.
   Consistent with total splenic B cells, no clear differences were
   observed in IgM expression within each subset in 17.8 and 11.2 T SMF
   compared with the sham groups (Figures [101]1J–M). These results
   suggested that ultra‐high SMF exposure might have a potential
   inhibitory effect on B cell differentiation.

FIGURE 1.

   FIGURE 1
   [102]Open in a new tab

   33.0 T ultra‐high static magnetic field interferes with the
   differentiation of peripheral B cells and inhibits antibody secretion.
   (A) Representative dot plots of peripheral marginal zone (MZ) B‐cell in
   splenic B cells from sham and 33.0 T SMF‐exposed groups. (B)
   Representative dot plots of peripheral B‐cell subsets including
   follicular (FO), transitional type‐1 (T1), and transitional type‐2 (T2)
   B cell in splenic B cells from sham and 33.0 T SMF‐exposed groups. (C)
   Representative dot plots of peripheral germinal center (GC) B‐cell in
   splenic B cells from sham and 33.0 T SMF‐exposed groups. (D–H) Shown
   are average percentages of MZ, FO, T1, T2, and GC B cells from sham and
   33.0 T/28.7 T/17.8 T/11.2 T SMF‐exposed groups. (I) IgM expression in
   splenic B cells from sham and 33.0 T/28.7 T/17.8 T/11.2 T SMF‐exposed
   groups. (J–M) Mean fluorescence intensity (MFI) of IgM in FO, MZ, T1,
   and T2 B cells from sham and 33.0 T/28.7 T/17.8 T/11.2 T SMF‐exposed
   groups. (N and O) The concentration of IgM/IgG in serum of mice in sham
   and 33.0 T SMF‐exposed groups (N = 5). (P and Q) The concentration of
   IgM/IgG in serum of mice in sham and 11.2T SMF‐exposed groups (N = 6).
   Sample were analyzed with FlowJo software, Error bars were shown as
   mean ± SEM. Dots represent individual mouse. *p < 0.05; **p < 0.01;
   ***p < 0.001 and ns: no statistical significance.

   We also measured the antibody content in serum to explore the effect of
   SMF on antibody secretion of B cells. IgM is the first antibody
   produced in the initial humoral immune response and is the vanguard of
   the infection resistance in the organism. Results showed that the
   concentration of IgM in the serum of mice in 33.0 T group was
   significantly lower than that in the sham group (Figure [103]1N). And
   IgG is the main antibody produced in the secondary humoral immune
   response, which is broadly distributed in the body and plays an
   important immune role. Similarly, 33.0 T exposure caused a decrease in
   IgG concentration in serum (Figure [104]1O). However, there were no
   significant changes in the concentration of either IgM or IgG after
   11.2 T SMF exposure (Figures [105]1P and [106]Q), as well as after 28.7
   and 17.8 T exposure (Figures [107]S2A–D), suggesting that the antibody
   secretion may be influenced by the intensity of the magnetic field.

   For other cell surface molecules (CD19, CD21, CD23, and IgD), most of
   them were normal either in total splenic B cells or in each subset
   under ultra‐high and high magnetic field exposures (Figures [108]2A–D
   and [109]S1A–P). Annexin V and Ki67 represent cell apoptosis and
   proliferation, respectively, and we did not observe any effects of SMF
   exposure with 4 different intensities on Annexin V and Ki67 expression
   in total splenic B cell as well as in each subset (Figures [110]2E–K).

FIGURE 2.

   FIGURE 2
   [111]Open in a new tab

   SMF exposure has little effect on peripheral B cell proliferation,
   apoptosis and the expression of CD19, IgD, CD23, and CD21 on B cells.
   (A–D) MFI of CD19, IgD, CD23, and CD21 in splenic B cells from sham and
   33.0 T/28.7 T/17.8 T/11.2 T SMF‐exposed groups. (E and F) Percentage of
   Annexin V and Ki67‐positive cells in total splenic B cell and FO, MZ,
   T1, T2, B subsets in sham and 33.0 T SMF‐exposed groups. (G–I)
   Percentage of Annexin V positive cells in total splenic B cell and FO,
   MZ, T1, T2, B subsets in sham and 28.7 T/17.8 T/11.2 T SMF‐exposed
   groups. (J and K) Percentage of Ki67‐positive cells in total splenic B
   cell and FO, MZ, T1, T2, B subsets in sham and 28.7 T/17.8 T
   SMF‐exposed groups. Samples were analyzed with FlowJo software, Error
   bars were shown as mean ± SEM. Dots represent individual mouse.
   *p < 0.05; **p < 0.01; ***p < 0.001 and ns: no statistical
   significance.

   Overall, exposure to ultra‐high SMF interferes with the differentiation
   and development of peripheral B cells and has an impact on some
   important cell surface molecules, such as IgM, which might influence
   cellular function. Meanwhile, 33.0 T SMF exposure also decreases the
   concentration of IgM and IgG and thus might weaken the humoral immune
   response. But the impacts of SMFs with different intensities on
   apoptosis and proliferation have not been found yet. In addition, in
   the high intensity magnetic field exposure groups (17.8 and 11.2 T), no
   clear difference was found from the sham groups in the above aspects,
   which might indicate that the inhibition of SMF on the differentiation
   of peripheral B cells and the expression of important surface molecules
   may be SMF intensity dependent, with a greater effect at higher
   magnetic field intensity.

2.2. 33.0 T SMF exposure suppresses proximal BCR signaling

   After B cells receive antigen stimulation, BCRs aggregate into clusters
   and recruit BCR signalosome associated molecules, which mediate the
   cascading BCR signaling and ultimately activate B cells. And BCR
   signaling plays a pivotal role in the differentiation and development
   of B cells. Thus, we used confocal microscopy (CFm) to continually
   observe the antigen internalization and presentation following BCR
   stimulation, as well as the participation and expression intensity of
   signaling molecules in the internalization process of B cell activation
   via colocalization analysis. The content of phosphotyrosine proteins
   (pY) could reflect the overall level of BCR signaling, and BTK is a
   crucial downstream molecule. Therefore, to investigate the impact of
   ultra‐high SMF on BCR signaling, we examined the spatiotemporal
   expression of pY and the activation of BTK in splenic B cells of mice
   in 33.0 T group and sham group with CFm at 0, 5, 10, and 30 min of
   soluble antigen (sAg) stimulation. We found noticeable decreases in the
   level of pBTK and pY in 33.0 T group after stimulation
   (Figures [112]3A, B, and [113]C). Along with this, compared with the
   sham group, the colocalization between pY and BCR was significantly
   reduced at 0 and 30 min (Figures [114]3A and [115]D), and the
   colocalization between pBTK and BCR was found to be reduced at 30 min
   of stimulation as well (Figure [116]3E). CD19 is a coreceptor of BCR,
   which has been known to be critical for B cell survival.[117] ^28
   Colocalization of pCD19 with BCR in exposure mice was significantly
   lower than in sham group at 10 min after sAg stimulation
   (Figures [118]3G and [119]K). Similarly, we also detected the
   activation of a negative regulator of proximal BCR signaling SHIP‐1. We
   found a decreased colocalization of pSHIP‐1 and BCR in the exposed
   group at 10 to 30 min following stimulation (Figures [120]3H and
   [121]L). Additionally, the levels of pCD19 and pSHIP were also
   decreased during 10 and 30 min (Figures [122]3I and [123]J). Meanwhile,
   the expression levels of pCD19 and pSHIP were much lower at 5 min after
   sAg stimulation in 33.0 T exposure group than those in the sham group
   (Figure [124]3F). All these results suggest that ultra‐high SMF
   exposure negatively regulates the spatiotemporal expression of key
   signaling molecules to restrain proximal BCR signaling.

FIGURE 3.

   FIGURE 3
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   33.0 T ultra‐high static magnetic field exposure suppresses proximal
   BCR signaling. Splenic B cells from sham and 33.0 T SMF‐treated mice
   were stimulated with AF594 F(ab′)2 goat anti‐mouse IgG + IgM (10 μg/mL)
   for 0, 5, or 10 min, then fixed, permeabilized, and stained for markers
   following. (A) Representative confocal microscopy (CFm) images of
   phosphotyrosine (pY), phosphorylated BTK (pBTK) and BCR (60× objective,
   scale bar = 2 μm). (B and C) CFm analysis of MFIs of pY and pBTK. (D)
   Colocalization between pY and BCR. (E) Colocalization between pBTK and
   BCR. The colocalization was analyzed using Pearson's correlation
   coefficient. (F) Western blotting of phosphorylated CD19 (pCD19) and
   phosphorylated SHIP (pSHIP) levels in splenic B cells activated
   biotin‐conjugated F(ab’)2‐anti‐mouse‐Ig (M + G) and streptavidin for 0,
   5, and 10 min. (G and H) Representative CFm images of pCD19 and pSHIP
   (60× objective, scale bar = 2 μm). (I and K) MFIs of pCD19 and
   colocalization between pCD19 and BCR. (J and L) MFIs of pSHIP and
   colocalization between pSHIP and BCR. *p < 0.05; **p < 0.01;
   ***p < 0.001; ****p < 0.0001 and ns: no statistical significance.

2.3. Decreased BCR clustering and signalosome recruitment in B cells from
33.0 T SMF‐exposed mice leads to the downregulation of BCR signaling

   The early events of B cell activation, including BCR clustering and
   signalosome recruitment, are crucial for the conduction of BCR
   downstream signaling. To investigate the influence of ultra‐high SMF on
   the early activation of B cells, we used TIRFm to assay the formation
   of BCR clusters and other events close to the membrane in 33.0 T and
   sham groups when membrane antigen (mAg) stimulated for 3, 5, and 7 min.
   The area of the cell contact zone indicated the spreading of B cells.
   Additionally, it was significantly smaller in the SMF‐exposed group
   than in the sham group at 5 min (Figures [126]4A, E, and [127]F).
   Moreover, the MFI of the BCR, which represented the aggregation of BCR,
   was reduced in 33.0 T exposure group at 5–7 min after stimulation
   (Figures [128]4A, D, and [129]F). For the overall level of BCR
   signaling, the MFI of pY reached the peak at 5 min, and it was lower in
   the cells of exposed mice at 5 min after mAg stimulation
   (Figures [130]4A and [131]B). We also found that the MFI values of pBtk
   and pSHIP in 33.0 T group were reduced compared with those of sham
   group at 5 min (Figures [132]4A, C, F, and [133]G). All these results
   suggest that ultra‐high SMF stimulation weakens BCR signaling
   conduction by inhibiting BCR clustering and signalosome recruitment.

FIGURE 4.

   FIGURE 4
   [134]Open in a new tab

   33.0 T ultra‐high static magnetic field exposure suppresses BCR
   clustering and decreases signalosome recruitment. (A) Representative
   total internal reflection fluorescence microscopy (TIRFm) images of pY
   and pBTK at 3, 5, or 7 min of activation with 10 μg/mL AF546 F(ab’)2
   anti‐mouse Ig (M+ G) (100× objective, scale bar = 2 μm). (B and C)
   Quantitative analysis of pY and pBTK MFI. (D and E) MFI of BCR and
   B‐cell contact area were quantified using NIS‐Elements AR 5.0.1
   software. (F) Representative TIRFm images of pSHIP (100× objective,
   scale bar = 2 μm). (G) Quantitative analysis of pSHIP MFI. *p < 0.05;
   **p < 0.01; ***p < 0.001; ****p < 0.0001 and ns: no statistical
   significance.

2.4. 33.0 TSMF stimulation impedes the accumulation of F‐actin

   Earlier studies have shown that the stimulation of SMF with low or
   moderate intensity affects the orientation, assembly and content of
   actin filaments.[135] ^29 , [136]^30 , [137]^31 And the reorganization
   of actin has been proven to exert a crucial role in B cell
   activation.[138] ^32 F‐actin is involved in receptor aggregation,
   signal molecule recruitment, and influencing BCR signaling positively
   or negatively. In addition to this, F‐actin also affects B cell
   adhesion and migration through controlling B cell morphology. Besides
   that, BCR signaling regulates F‐actin mediated by WASp.[139] ^27 Thus,
   to explore the effect of SMF with ultra‐high intensity on B cell actin
   cytoskeleton, we detected the levels of activated WASp and F‐actin as
   well as their colocalization with BCR via CFm at different time points
   after sAg stimulation. Colocalization between F‐actin and BCR was
   significantly lower in 33.0 T exposure group at 10 min after
   stimulation than that in the sham group (Figures [140]5A and [141]C).
   Meanwhile, the level of F‐actin in B cells of 33.0 T group declined
   obviously after 30 min of stimulation (Figures [142]5A and [143]B).
   Colocalization of pWASp and BCR was less in the 33.0 T group compared
   with that in the sham group at 10 min (Figures [144]5A and [145]E), and
   the MFI of pWASp was also decreased markedly from 10 to 30 min after
   stimulation (Figures [146]5A and [147]D). We conducted another assay
   using TIRFm with higher resolution. The data show that the content of
   F‐actin in the contact area of B cells in the 33.0 T group was reduced
   at 5–7 min after the treatment of B cells with mAg (Figures [148]5G and
   [149]H). Dock8 regulates actin accumulation by means of WASp,[150] ^33
   and studies have proven that Mst1 and WASp regulate each other through
   indirect effects.[151] ^34 Interestingly, we noticed that the
   expression of pWASp in 33.0 T group was decreased at 5 min compared
   with sham group (Figure [152]5F). The expressions of pMst1 and Dock8 in
   33.0 T group were lower than that in sham group at 5 and 10 min after
   stimulation (Figure [153]5F). This indicates that 33.0 T SMF exposure
   has an inhibitory effect on Mst1–Dock8–WASp axis. Altogether, the above
   results suggest that ultra‐high SMF exposure decreases F‐actin
   accumulation and that the potential mechanism for this process may be
   due to the repression of the Mst1–Dock8–WASp axis in the downstream of
   BCR singling caused by SMF exposure.

FIGURE 5.

   FIGURE 5
   [154]Open in a new tab

   33.0 T ultra‐high static magnetic field stimulation impedes the
   accumulation of F‐actin. (A) Representative confocal microscopy (CFm)
   images of phosphorylated WASP (pWASP) and F‐actin (60× objective, scale
   bar = 2 μm). (B and C) MFIs of F‐actin and colocalization between
   F‐actin and BCR. (D and E) MFIs of pWASP and colocalization between
   pWASP and BCR. (F) Western blotting of pWASP, pMST1, and DOCK8 levels
   in splenic B cells with F(ab’)2 activation. (G) Representative total
   internal reflection fluorescence microscopy (TIRFm) images of F‐actin
   at 3, 5, or 7 min of activation with 10 μg/mL AF546 F(ab’)2 anti‐mouse
   Ig (M+ G) (100× objective, scale bar = 2 μm). (H) MFI of F‐actin in the
   contact zone. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 and
   ns: no statistical significance.

2.5. Exposure to SMFs affect the transcriptome profile of B cells and
disturbs metabolism‐related molecules

   To investigate the possible mechanisms for the effects of SMF
   stimulation on B cell function, we performed transcriptome sequencing
   and KEGG analysis of B cells from 11.2 T SMF exposure group and sham
   group. Compared with the sham group mice, a total of 305 upregulated
   transcripts and 253 downregulated transcripts were detected in 11.2 T
   SMF‐treated mice B cells (Figure [155]6A). We tagged the top 100
   differential genes with padj less than 0.001 (Figure [156]6B). To
   better understand the impact of SMF stimulation, we enriched the
   differentially expressed genes to 14 KEGG pathways, where p values
   indicate the degree of significant enrichment (Figure [157]6C). We
   found high enrichment in immune‐related pathways, such as B cell and T
   cell receptor signaling pathways and Th1 and Th2 cell differentiation,
   which strongly affected the regulation of humoral and cellular
   immunity. NF‐kappa B signaling pathway regulates genes involved in
   immunity, inflammation, and cell survival. Some other important
   intracellular signaling pathways were also highly enriched, especially
   FoxO, mTOR, and PI3K–Akt signaling pathways, and they perform essential
   regulatory roles in cell growth and metabolism, cytoskeleton, and other
   processes. Moreover, some fundamental cellular processes, such as
   autophagy, were also enriched. As for metabolic pathways, oxidative
   phosphorylation, fatty acid, and pyruvate metabolism were enriched to
   some extent. These results suggested that 11.2 T magnetic field
   exposure may affect the immune system in diverse ways, including cell
   differentiation, metabolism, and important signaling pathways. Earlier
   sequencing studies on human embryonic lung fibroblasts under 3.0 T
   magnetic field exposure found no obvious difference in protein‐gene
   expression between experimental and control groups,[158] ^35 which also
   indicated that the effect of SMF might be intensity dependent. Since we
   sequenced transcriptome 2 months after the treatment of mice, this is
   an indication of a possible long‐term influence of high SMF on the
   transcriptional profile of B cells.

FIGURE 6.

   FIGURE 6
   [159]Open in a new tab

   Static magnetic fields exposure impacts cell metabolism. (A) Heat map
   of differential transcripts between sham and 11.2 T SMF‐exposed groups.
   (B) Volcano plot of differentially expressed mRNAs, marking the top 100
   differential genes with padj < 0.001. Blue dots represent downregulated
   genes (padj < 0.05 and |log 2 (FoldChange)| ≤ −1) and orange represents
   upregulated genes (padj < 0.05 and |log 2 (FoldChange)| ≥ 1). (C)
   Significantly differential transcripts enriched to 14 KEGG signaling
   pathways. (D) Western blotting of pS6, pAKT, and pFOXO1 levels in
   splenic B cells activated biotin‐conjugated F(ab’)2‐anti‐mouse‐Ig (M +
   G) and streptavidin for 0, 5, and 10 min in splenic B cells from sham
   and 33.0 T SMF‐exposed groups.

   To further verify the effect of SMF on B cell metabolism, we detected
   the effect of 33.0 T ultra‐high SMF stimulation on the metabolic
   signaling pathway of splenic B cells. We lysed splenic B cells after
   sAg stimulation and analyzed the content of metabolism‐related
   molecules with western blotting. PI3K–Akt–mTORC1 is an essential
   metabolic signaling pathway in B cells, and Akt is a key regulator in
   it. The outcome showed that the expression of pAkt in 33.0 T group was
   significantly lower than the sham group at 5 min (Figure [160]6D). S6
   is the downstream molecule of mTORC1 related to protein synthesis, and
   FOXO1 is a glucose metabolism‐related transcription factor in BCR
   distal signaling. The levels of pS6 and pFOXO1 in 33.0 T group were
   also decreased at 5 min (Figure [161]6D). Therefore, 33.0 T SMF
   exposure may affect B cell metabolism.

   The above results indicate that both high and ultra‐high SMF
   stimulation could affect B cell metabolism to a certain degree,
   especially after 33.0 T stimulation, the metabolic signals were
   significantly reduced. SMF alters B cell homeostasis and makes B cell
   immunosuppressive by interfering with cellular metabolism, but this
   still needs to be further verified.

3. DISCUSSION

   The field strength of SMFs in MRI has been enhanced significantly in
   the past few decades for better imaging resolution and detection
   capability. As a widely used technique in medicine, the effects of SMF
   on organisms has also been extensively studied, but its effects on B
   cells has not been explored. In this study, we first established the
   effects of magnetic fields on humoral immunity. Due to the technical
   limitations of magnetic field intensity, previous studies on the
   biological effects of SMF have mainly focused on low and moderate
   intensities. Earlier studies have found that SMF could affect natural
   immune cells, for instance, promoting M2 polarization of
   macrophages.[162] ^18 , [163]^19 Furthermore, studies on the roles of
   SMF on adaptive immune cells concentrated on T cells and revealed that
   SMF could affect the number of stimulated CD4+ T cells and CD8+ T cells
   and the secretion of cytokines.[164] ^20 , [165]^23 , [166]^24 It has
   also been known the implications of SMF on the balance between pro‐ and
   anti‐inflammatory molecules[167] ^20 and its potential antitumor
   activity.[168] ^36 A previous study has shown a significant reduction
   in spleen size in mice after 2 months of 33.0 T SMF exposure,[169] ^12
   which suggested that ultra‐high magnetic field might have a long‐term
   effect on immune organs, but its effects on humoral immunity have not
   been further explored. As a widely used technique in medicine, the
   effect of SMF on organisms has also been extensively studied, but its
   effect on B cells has not been explored. In this study, we first
   established the effects of magnetic fields on humoral immunity, filling
   the blank in the field of side effects of magnetic fields on B cells.

   Meanwhile, our group has published several articles about long‐term
   treatment with magnetic fields and found that SMFs do not have many
   safety issues for healthy mice. For example, we exposed healthy mice
   continuously to 0.1–0.2 T SMF for 1.7 years and did not reveal harmful
   effects.[170] ^37 We also found that high SMFs of 9.4 T exposure for
   200 h did not have observable defects in tumor‐bearing mice.[171] ^38
   However, 14 h of prolonged treatment of gradient high SMFs had negative
   effects on type 1 and 2 diabetes mice, including spleen, hepatic, and
   renal tissue impairment.[172] ^39 We have summarized the effect of
   long‐term SMF exposure in a book chapter recently, which can provide
   more information on this topic.[173] ^40 The main purpose of this
   article is to investigate the effects of ultra‐high magnetic fields up
   to 33.0 T, hoping to provide information for the future development of
   high‐field MRI and other related devices, so our study was conducted at
   SMFs with four high or ultra‐high intensities, responding to the
   current demands for higher intensity magnetic fields in medical
   applications. Considering that the current clinical MRI detection time
   is about 0.5–1 h, 1‐h magnetic field treatment is used as the
   experimental condition here. And the sham groups were designed to
   exclude the influence of irrelevant factors, such as the environment
   and noise produced by the magnetic field‐generating equipment.

   Our results revealed that ultra‐high SMF exposure could cause
   immunosuppression. 28.7 and 33.0 T magnetic resulted in abnormal B
   cells peripheral growth and differentiation, such as decreases in the
   proportion of T2 subset and mIgM expression. And 33.0 T exposure
   reduced the concentration of IgM and IgG in serum, which might cause a
   weakened humoral immune response. These effects were intensity
   dependent. We also explored the changes in BCR signaling and F‐actin in
   the splenic B cells of 33.0 T group after antigen stimulation for the
   detailed mechanisms. Exposure to ultra‐high magnetic field decreased
   BCR clustering and proximal positive and negative signalosome
   aggregation after stimulation, which suppressed the conduction of BCR
   signaling. Humoral immune response demands optimal BCR signaling, and
   both higher and lower BCR signaling may cause reduced response.[174]
   ^41 , [175]^42 The α‐helix structure has been proved to give the
   protein diamagnetic anisotropy.[176] ^43 In humans, the structure of
   the IgM–BCR complex was resolved by cryo‐EM, which revealed that the
   transmembrane region of mIgM and Igα/Igβ formed a four‐helix bundle
   that appeared to be conserved among all BCR isoforms.[177] ^44 This may
   be related to the inhibition of BCR aggregation by ultra‐high intensity
   magnetic fields. Similar to previous studies, F‐actin accumulation was
   reduced after magnetic field exposure, as it can form organized
   polymers, which have strong antimagnetic anisotropy. Besides, the
   activation of Mst1–Dock8–WASp axis‐related molecules was depressed
   after antigen stimulation in 33.0 T group, probably leading to reduced
   accumulation of F‐actin. Thus, we speculate that ultra‐high SMF
   directly acts on the BCR complex and F‐actin to disrupt the activity of
   them, and that the interaction between BCR signaling and F‐actin
   aggregation may also be involved in this process. However, although
   more detailed mechanisms need to be further explored, our data here
   show that ultra‐high SMF causes immunosuppression by inhibiting BCR
   signaling and the peripheral differentiation of B cells.

   Transcriptome analysis highlighted the alteration in BCR signaling and
   other important intracellular signaling pathways, further confirming
   our experiments of mice model. On the other hand, the enrichment of
   metabolic pathways suggested that SMF might affect B cell metabolism,
   as confirmed by the immunoblotting experiment we performed later, which
   found that SMF exposure inhibits the activation of PI3K–Akt
   axis‐related molecules. Magnetic fields have been shown to impact
   glucose and lipid metabolism in mice, such as downward moderate SMF
   treatment resulted in lower blood cholesterol and glucose levels and
   reduced body weight and lipid accumulation in the liver in type 2
   diabetic mouse models,[178] ^45 while 33.0 T SMF cause a 24.3% increase
   in cholesterol in exposure group compared with the sham group.[179] ^12
   Other studies have come to different or even opposite conclusions,[180]
   ^46 , [181]^47 pointing to the fact that the effect of magnetic on
   metabolism was influenced by magnetic field parameters including
   intensity and direction. Moreover, SMF may also prevent the progression
   of high‐fat‐diet‐induced diabetes to some extent.[182] ^45 Research
   over the past decade has demonstrated that B cells play a central role
   in chronic inflammation and immune dysregulation associated with
   obesity, while immune cell interaction and inflammation in the adipose
   tissue regulate systemic glucose and fatty acid metabolism.[183] ^48
   The PI3K–Akt signaling pathway promotes B cell anabolism and
   activation.[184] ^49 However, it remains unclear whether impaired B
   cell metabolism affects adipose tissue and thus alters organism
   metabolism. Therefore, obesity models or type 2 diabetes models can be
   induced and further studied.

   In conclusion, ultra‐high SMF exposure leads to immunosuppression,
   reduces serum concentration of IgG and IgM, interferes with peripheral
   B cell development and signaling, and has some inhibition of metabolic
   signaling pathways. More studies exploring the effects of different
   magnetic field parameters, including direction, intensity, gradient,
   and exposure, are expected. Although magnetic field is an effective
   medical treatment, attention should be paid to the side effects of
   ultra‐high SMF on the organism, and the best combination of magnetic
   field parameters should be selected.

   However, the main limitation of this study is that we could not obtain
   data with a larger sample size. Due to the very high cost of
   electricity for performing these ultra‐high magnetic field experiments
   in the water‐cooled magnet, the small‐bore size of the ultra‐high
   magnet, as well as the very limited machine time available for these
   ultra‐high magnets, there are very few reports about >20 T ultra‐high
   magnetic field experiments on animals. It is more difficult to achieve
   large samples, especially for research on SMF above 30 T. Due to the
   failure of library construction, we did not obtain sufficient
   ultra‐high magnetic field sequencing data. We sequenced only one 28.7 T
   SMF‐treated mouse (data not shown). In order to verify the findings
   from 11.2 T SMF‐treated mouse, we measured mitochondrial function and
   the content of the key molecules in PI3K–Akt–mTORC1 pathway to further
   investigate the metabolic effects of ultra‐high magnetic field
   exposure. Although the results indicated that the metabolic signaling
   of B cells was impaired after high and ultra‐high magnetic field
   stimulation, the possibility that SMF stimulation may impact B cell
   function by impairing cellular metabolism still needs to be further
   verified. In addition, we seldom explore the influence of magnetic
   fields on the physiological functions of B cells, such as cytokine
   secretion and specific antibody production, which remain to be further
   investigated to help us better understand the effects of ultra‐high SMF
   exposure on B cells.

4. MATERIALS AND METHODS

4.1. SMF exposure system

   The water‐cooled magnet (WM#5) of the National Major Scientific and
   Technological Infrastructure Construction Project in China, Chinese
   Academy of Sciences, Hefei, was used. The SMF exposure device consists
   of three parts. The inner part is a cylinder stacked with eight
   nonmagnetic stainless tubes, each containing one mouse. The middle is a
   precise temperature controlling and monitoring equipment with a double
   layer. And the outer is a water‐cooled magnet to provide SMF. Moreover,
   sham‐exposed groups were treated in the same environment as their
   corresponding SMF‐exposed groups. Detailed assembly and parameters have
   been described previously.[185] ^12

4.2. Mice

   Forty‐six healthy 8‐week‐old male C57BL/6 mice were placed on four
   different layers in the water‐cooled magnet randomly. The magnetic
   field intensity of each layer is 33.0, 28.7, 17.8, and 11.2 T
   respectively, with an upward magnetic field gradient. For the mice in
   each layer, they were divided into sham‐exposed group (N = 5 for 33.0 T
   sham group and N = 6 for the other groups) and SMF‐exposed group (N = 5
   for 33.0 T group and N = 6 for the other groups). We exposed mice to
   the device for 70 min as described in the previous experiment.[186] ^12
   For each group, six mice in total were treated in six independent
   experiments over 3 days. Two months after the exposure, the spleens of
   mice were taken for isolating and analyzing B cells. During the
   experiment, the mice were housed in a standard barrier environment with
   a 12‐h light/dark cycle and ad libitum feeding.

4.3. ELISA

   Serum was collected from the mice of SMF‐exposed groups and their
   corresponding sham‐exposed groups. The QuantiCyto® Mouse IgM ELISA kit
   (EMC 129; NeoBioscience) and Mouse IgG (Total) ELISA Kit (RK00375;
   abclonal) were used to measure the concentration of IgM and IgG in
   serum in accordance with the experimental protocol provided by
   manufacturer.

4.4. Cell isolation

   Mononuclear cells were isolated from mouse spleen by gradient
   centrifugation in Ficoll solution (17‐1440‐02; GE Healthcare). Then
   splenic B cells were purified by incubation with anti‐CD90.2 mAb
   (105310; BioLegend) and guinea pig complement (C300‐0500; Rockland) for
   30 min. In order to further remove adherent cells, the cells were
   cultured in the incubator (37°C, 5% CO[2]) for 1 h.

4.5. Flow cytometry

   For the analysis of splenic B cell subsets, cells were stained with
   different surface markers. The whole operation was kept on ice and out
   of light. The antibodies used are as follows: APC‐anti‐CD21 (123412;
   BioLegend), BV421‐anti‐IgM (406518; BioLegend), BV510‐anti‐B220
   (103234; BioLegend), FITC‐anti‐CD19 (101506; BioLegend),
   PerCP/Cy5.5‐anti‐IgD (405710; BioLegend), FITC‐anti‐CD95 (152606;
   BioLegend), AF647‐anti‐GL7 (144606; BioLegend), percp‐7AAD (6084701; BD
   Pharmingen), and PE‐anti‐CD23 (101608; BioLegend). In addition,
   PE‐Cy7‐anti‐ki67 (1983210; Invitrogen) was used to label cells in the
   proliferation cycle and Annexin V (640906; BioLegend) was used to
   detect apoptosis. Data were acquired on Attune NxT (Thermo Fisher) and
   analyzed by the FlowJo software (TreeStar).

4.6. CFm and TIRFm

   For confocal analysis, purified splenic B cell suspension was dropped
   onto the poly‐lysine coated slides and incubated at 37°C for 30 min to
   make cells adherent. After stimulating cells in a water bath (37°C)
   with Alexa Fluor 594 F(ab ')2 Goat Anti‐mouse IgM+IgG (H+L)
   (115586‐068; Jackson ImmunoResearch), cells were fixed with 4%
   paraformaldehyde at 0 min, 5 min, 10 and 30 min, respectively. Next,
   cells were permeabilized with 0.05% saponin buffer (S4521‐10G;
   Sigma–Aldrich), and stained with primary and secondary antibodies
   successively. All the operations were performed on ice and protected
   from light. Finally, pictures were taken with Nikon Eclipse Ti‐PFS and
   analyzed with NIS‐elements AR 5.01 software for MFI and the correlation
   coefficient of colocalization. Reagents used are listed below:
   anti‐phosphotyrosine (pY, 05−321; Merck Millipore), anti‐pSHIP1 (3941S;
   Cell Signaling Technology), anti‐pCD19 (ab63443, Abcam), anti‐pBtk
   (ab52192; Abcam), anti‐pWASp (A300‐205A; Bethyl Laboratories),
   ActinGreen 488 ReadyProbes ([187]R37110; Thermo Fisher), Alexa Fluor
   405 goat anti‐mouse IgG (A‐31553; Thermo Fisher), Alexa Fluor 488 goat
   anti‐rabbit IgG (A‐11008; Thermo Fisher), and Alexa Fluor 405 goat
   anti‐rabbit IgG (A‐31556; Thermo Fisher).

   For TIRFm analysis, as described previously,[188] ^50 , [189]^51
   splenic B cells from 33.0 T exposure group and sham group were
   incubated with Ag‐tethered lipid bilayer and activated in water bath
   (37°C) for 3, 5, and 7 min, respectively. After fixation and
   permeabilization, cells were stained with pY, pBtk, pSHIP, F‐actin, and
   pWASp. Images were taken by TIRFm (Nikon Eclipse Ti‐PFS). For
   image‐related analysis, MFI and area of contact zone were detected
   using NIS‐elements AR 5.01 and background fluorescence was subtracted
   in the measurement of MFI. There were over 50 individual cells analyzed
   for each set of data.

4.7. Western blotting

   Isolated splenic B cells from the 33.0 T group and sham group were
   incubated with Biotin‐Fab’ of anti‐mouse Ig(M+G) on ice for 30 min,
   followed by continued incubation with streptavidin for 10 min. Cells
   were then activated at 37°C for the indicated time. After lysing cells
   with RIPA buffer (P0013B; Beyotime), the cell lysates were separated by
   SDS‐PAGE and protein bands were loaded onto nitrocellulose membranes
   through electrophoretic transfer. After blocking the blank sites,
   membranes were processed with anti‐pCD19 (ab63443; Abcam), anti‐pSHIP
   (3941S; CST), anti‐pS6 (4856S; CST), anti‐pAkt (4060L; CST),
   anti‐pFOXO‐1 (9461S; CST), anti‐pWASp (A300‐205A; Bethyl Laboratories),
   anti‐pMST1 (3681S; CST), anti‐DOCK8 (11622‐1‐AP; Proteintech), and the
   secondary antibodies were added later. Images were captured from
   ChemiDoc™XRS+ imaging systems (Bio‐Rad). β‐Actin (60008‐1‐IG‐10;
   Proteintech) was used as a loading control.

4.8. Transcriptome sequencing and data analysis

   Transcriptome libraries were constructed using Hieff NGS® Ultima
   Dual‐mode mRNA Library Prep Kit for Illumina® (Yeasen; 12252ES96)
   following the manufacturer's instructions. mRNA libraries were
   sequenced on GenoLab M platform (GeneMind Biosciences LTD.) using a
   150‐cycle paired‐end high‐output sequencing mode.

   Raw sequencing data (Fastq) were processed through a GeneMind in‐house
   perl pipeline. After filtering adapter, ploy‐N or low‐quality reads,
   clean reads were obtained and mapped to the reference genome sequence
   (mm10/GRCm39.primary) via HISAT2 tools software. Expression
   quantification and differential expression were performed using
   StringTie and DESEq2. Pathway enrichment analysis (KEGG) was conducted
   with the “clusterProfiler” R package.

   The RNA‐seq data sets presented in this study can be found in online
   repository of China National GeneBank DataBase (CNGBdb) with accession
   number CNP0004667.

4.9. Statical analysis

   Unpaired two‐tailed Student's t‐test was used for comparisons between
   two groups. GraphPad Prism 8 Software was used to assess statical
   significance. Experimental values were shown as mean ± standard error
   of mean (SEM). Statistical significance was set at p < 0.05.
   Statistical significance differences defined as *p < 0.05, **p < 0.01,
   ***p < 0.001, and ****p < 0.0001.

AUTHOR CONTRIBUTION

   H. G., L. L., and M. X. performed the flow cytometry experiments. H.G.
   carried out the western blotting. D. K. performed the confocal
   experiments. Q. C and Y. J. performed the TIRFm experiments. B. Y.
   established SMF and sham‐exposed mouse models. H. G., X. Z., and J. L.
   carried out transcriptome sequencing and analysis. H. G. analyzed the
   data and generated figures. Y. F. drafted the manuscript. C. L., X. Z.,
   G. H., B. Y., F. G., H. F., J. S., L. Y., J. L., and X. D. reviewed and
   revised the manuscript. X. Z. and C. L. designed and supervised the
   study. All authors approved the final paper as submitted and agreed to
   be accountable for all aspects of the work.

CONFLICT OF INTEREST STATEMENT

   Authors Xin Zhang and Juan Lai were employed by GeneMind Biosciences
   Company Limited, Shenzhen, China. The remaining authors declare that
   the research was conducted in the absence of any commercial or
   financial relationships.

ETHICS STATEMENT

   The animal protocols were approved by the ethical and humane committee
   of Hefei Institutes of Physical Science, Chinese Academy of Sciences
   and carried out strictly in accordance with the related regulations.
   (Ethics No. SWYX‐DW‐2021‐22).

Supporting information

   Supporting Information
   [190]Click here for additional data file.^ (492.8KB, docx)

ACKNOWLEDGMENTS