Deletion of Mettl3 at the Pro-B Stage Marginally Affects B Cell Development and Profibrogenic Activity of B Cells in Liver Fibrosis

N6-methyladenosine (m6A) modification plays a pivotal role in cell fate determination. Previous studies show that eliminating m6A using Mb1-Cre dramatically impairs B cell development. However, whether disturbing m6A modification at later stages affects B cell development and function remains elusive. Here, we deleted m6A methyltransferase Mettl3 from the pro-B stage on using Cd19-Cre (Mettl3 cKO) and found that the frequency of total B cells in peripheral blood, peritoneal cavity, and liver is comparable between Mettl3 cKO mice and wild-type (WT) littermates, while the percentage of whole splenic B cells slightly increases in Mettl3 cKO individuals. The proportion of pre-pro-B, pro-B, pre-B, immature, and mature B cells in the bone marrow were minimally affected. Loss of Mettl3 resulted in increased apoptosis but barely affected B cells' proliferation and IgG production upon LPS, CD40L, anti-IgM, or TNF-α stimulation. Different stimuli had different effects on B cell activation. In addition, B cell-specific Mettl3 knockout had no influence on the pro-fibrogenic activity of B cells in liver fibrosis, evidenced by comparable fibrosis in carbon tetrachloride- (CCl4-) treated Mettl3 cKO mice and WT controls. In summary, our study demonstrated that deletion of Mettl3 from the pro-B stage on has minimal effects on B cell development and function, as well as profibrogenic activity of B cells in liver fibrosis, revealing a stage-specific dependence on Mettl3-mediated m6A of B cell development.


Introduction
The development and maturation of B cells are tightly regulated processes that involve several steps [1][2][3]. First, bone marrow resident common lymphoid progenitor cells (CLPs) commit to the B cell lineage and enter the pro-B cell stage under the control of crucial transcription factors E2A, EBF, and Pax5 [4]. Then, heavy-chain DJ and VDJ rearrangements of immunoglobulin-gene happen in pro-B cells. When they start to express light-chain, the pro-B cells progress to the pre-B stage [5][6][7]. Once finishing VJ rearrangement of the light chain and expressing IgM, the cells become immature B cells. Then, the immature B cells undergo further negative selection, and the survival cells upregulate the B cell-activating factor receptor (BAFF-R) and acquire survival signals from BAFF. The survival signals support their survival when they exit bone marrow, enter the circulation, and migrate to the spleen for further maturation [4,8,9]. Growing evidence indicates that B cell differentiation is controlled by complex epigenetic and transcriptional programs [10][11][12][13].
However, the role of Mettl3-mediated m 6 A modification in B cell development and functions remains elusive. Zheng and colleagues found that Mb1-Cre-mediated ablation of Mettl14 resulted in the block of pro-B cell proliferation, pro-B to large pre-B, and large pre-B to small pre-B transition [41]. Mb1-Cre-mediated Mettl3 knockout showed a similar phenotype [42]. Since Mb1-Cre starts to express at the earliest pre-pro-B cell (CD19 − B220 mid Igκ/λ − Cd43 hi ) stage [41], whether knocking out Mettl3/Mettl14 at later stages affects B cell development and function is still unknown. Recently, Grenov et al. reported that Mettl3mediated m 6 A modification was required for the germinal center formation and maintenance [43].
Here, we deleted Metllt3 using Cd19-Cre (expressing from the pro-B cell stage on [44,45]) (Mettl3 cKO) to see the role of Mettl3-mediated m 6 A in later stage development and function of B cells. No developmental defects of Mettl3 cKO mice were observed. The frequency of total B cells in peripheral blood, peritoneal cavity, and liver, as well as B cell subsets at different developmental stages (pre-pro-B, pro-B, pre-B, immature B, and mature B cells) in the bone marrow, was comparable between Mettl3 cKO mice and wild-type control (WT) littermates, consistent with previous reports by Grenov et al. [43]. Deletion of Mettl3-mediated m 6 A using Cd19-Cre did not affect B cell proliferation and IgG production but promoted apoptosis in vitro. Moreover, different stimuli (LPS, CD40L, anti-IgM, or TNF-α) had different effects on B cell activation. As B cell contributes to hepatic fibrosis in an antibody-independent way [46], and Mettl3 was increased in B cells from fibrotic livers in published datasets [47], we explored the function of Mettl3 on B cells invivoby using CCl 4 -induced liver fibrosis model. The results showed that Mettl3 deletion in B cells does not affect liver fibrosis progression. Our study demonstrated that Mettl3 marginally affects Cd19 + B cell development, activation, and profibrogenic function in liver fibrosis.

Methods
2.1. Mice. Mettl3 flox/flox mice (kindly gifted by Professor Qi Zhou [48]) were crossed with Cd19-Cre mice (purchased from GemPharmatech Co. Ltd, Nanjing, China) to generate Mettl3 flox/flox /Cd19-Cre (Mettl3 cKO) mice. Mettl3 flox/flox littermates were used as WT controls. 6 to 8 weeks old sexand age-matched mice were used in this study. All mice were maintained on a C57BL/6 background and housed in specific pathogen-free conditions. Animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Third Affiliated Hospital of Sun Yat-sen University. Primers used for genotyping were listed in Supplementary Table 1. 2.2. CCl 4 -Induced Liver Fibrosis. CCl 4 (289116, Sigma-Aldrich, USA) was diluted with corn oil (O815211, Macklin, China) at a ratio of 1 : 4 and injected intraperitoneally (i.p.) into mice at 5 μl/g body weight twice per week for six weeks as described previously [49]. Samples were collected 24 hours after the last CCl 4 treatment.

RNA Isolation and Quantitative
Real-Time PCR (qRT-PCR). Total RNA was extracted with TRIzol reagent (15596026, Invitrogen, USA), followed by reverse transcription with the Fast All-in-One RT Kit (RT001, ES Science, China). cDNA was used as the template in real-time PCR with SYBR Green (4707516001, Roche, Switzerland). All reactions were performed in triplicates, and Gapdh was used as the internal control. The relative mRNA abundance was calculated using the ΔΔCt methods. Primers were listed in Supplementary Table 2. 2.5. Western Blot. Cell pellets or tissues were lysed, and protein concentration was detected by the BCA method. The proteins were equally loaded to SDS-PAGE gel, transferred onto nitrocellulose membranes, and then incubated sequentially with primary and second antibodies. The protein bands were developed by Chemidoc Imaging System (Biorad®) using Immobilon ECL Ultra Western HRP 2.8. Liver Lymphocyte Isolation. The liver was cannulated with a 25-gauge needle through the portal vein and perfused with 10 ml of ice-cold PBS. After removing the gall bladder, the liver was cut into segments and digested with 0.02% collagenase IV (C5138, Sigma-Aldrich, USA, 5 ml per liver) for 45 mins at 37°C on a shaker at the speed of 70 rpm. The liver slurry was centrifuged for 3 mins at 30 g. The supernatants were passed through a 70 μm mesh cell strainer (352350, BD Falcon, USA) and then centrifuged for 10 mins at 300 g at 4°C. The cell pellets were resuspended in 5 ml of mouse 1 × lymphocyte separation medium (7211011, DAKEWEI, China), overlaid by 0.5 ml RPMI-1640, then centrifuged at 800 g for 30 minutes at 4°C with no brakes. Lymphocytes at the interface were harvested, washed with RPMI-1640 supplemented with 5% fetal bovine serum (FBS, FSP500, ExCell, China), and used for further analyses.
2.9. Isolation of Lymphocytes from Peritoneal Cavity, Spleen, Blood, and Bone Marrow. Mice were anesthetized and exposed abdominal cavity. Sterilized PBS was used to wash the peritoneal cavity, and then the peritoneal lavage fluid was collected and centrifuged at 300 g for 5 mins at 4°C. Cells obtained were used for further analysis. Spleens were minced through a nylon mesh (Cell Strainer, 352340, BD Falcon, USA) to obtain single-cell suspensions in RPMI-1640 containing 5% FBS. Erythrocytes were lysed by incubating in RBC lysis buffer (140 mM NH4Cl, 17 mM Tris-HCl, and pH 7.65) for 3 minutes on ice. Peripheral blood was collected in EDTA-containing tubes, then underlaid with Ficoll-Paque™ PLUS (17-1440-02, GE Healthcare, USA), and centrifuged at 1000 g at room temperature for 20 minutes with no brakes. Lymphocytes were collected from the interface. Femur and tibia bones were used to isolate bone marrowderived lymphocytes. Both ends of the bone were carefully cut with sharp dissecting scissors. Bone marrow cells were flushed using PBS and then centrifuged at 300 g for 5 mins at 4°C. The cell pellets were resuspended in 1 ml RBC lysis buffer and lysed for 3 mins on ice. 5 volume of PBS was added to stop the reaction. Lymphocytes were collected by centrifugation at 300 g at 4°C, washed twice with RPMI-1640 supplemented with 5% FBS, and used for further analyses. , and corresponding isotype control antibodies were purchased from Biolegend. Antibodies were listed in Supplementary Table 3.

Generation of Cd19-Cre-Mediated B Cell-Specific Mettl3
Knockout Mice. By knocking out Mettl3 or Mettl14 using Mb1-Cre (starts to express at the earliest pre-pro-B cells), previous studies showed that m 6 A plays an essential role in early B cell development [41,42]. To investigate the role of (a) Relative mRNA expression   [44,45]. Genotype was monitored by genomic PCR of mouse tails (Figure 1(c)). To further confirm B cell-specific Mettl3 knockout, we sorted splenic CD19 + B cells and CD19cells from WT and Mettl3 cKO mice and conducted genomic PCR, western blot, and RT-qPCR for Mettl3 (Figures 1(d)-1(f)). The results showed specific and efficient knockout of Mettl3 occurred only in CD19 + B cells.  Figure 1A & 1B). Mice were born at expected Mendelian frequency, and no infection or other discernable differences were observed during regular feeding (data not shown). Interestingly, the spleen weight and the ratio of spleen weight to body weight were slightly increased in Mettl3 cKO groups (Figure 2(c) and Supplementary Figure 1C). Deletion of Mettl3 or Mettl14 with Mb1-Cre resulted in a significant decrease of B cells in the peripheral and even disappeared in the spleen and peritoneal cavity [41,42]. We determined B cell percentage in peripheral blood, peritoneal cavity, liver, and spleen and found that there was no significant difference in CD19 + B cell fraction between WT and Mettl3 cKO mice in peripheral blood, peritoneal cavity, and liver (Figures 2(d)-2(f) and Supplementary  Figures 2A-2D). However, the proportion of CD19 + B cells in the spleen was slightly but significantly increased in Mettl3 cKO individuals (Figure 2(g)), which may contribute to the increased spleen weight of Mettl3 cKO mice (Figure 2(c) and Supplementary Figure 1C). Histological analysis with H&E staining also showed no structural and histological     Figure 2E). Whole bone marrow cells were segregated based on B220 and CD43 expression (Figure 3(c), left) and divided B cell precursors into B220 + CD43 + progenitor cells based on CD24 and BP-1 expression (Figure 3(c), center) and more mature B220 + CD43 − populations based on surface IgM and IgD expression (Figure 3(c), right) [50,51]. Both CD43 + populations (containing the most immature B-cell populations in the marrow) and CD43populations (mainly containing pre-B-cells, immature and mature B cells) were comparable between WT and Mettl3 cKO littermates (Figures 3(d) and 3(e)). Moreover, there is no significant difference in the proportion of pre-pro-B (fraction (Fr.) A, B220 + CD43 + CD24 -), early pro-B (Fr. B, B220 + CD43 + CD24 + BP1 -), and late pro-B (Fr. C, B220 + CD43 + CD24 + BP1 + ) fractions in the bone marrow between Mettl3 cKO mice and WT controls (Figure 3(d)). We also observed a minimal difference of IgM + IgDimmature B cells and IgM + IgD + mature B cells in B220 + CD43 − fractions (Figure 3(e)). Therefore, the knockout of Mettl3 in B cells with Cd19-Cre barely influenced B cell development and maturation.

Loss of Mettl3 Has Minimal Effects on B Cell Activation and Proliferation but Promotes Apoptosis upon Stimulation
In Vitro. To investigate whether Mettl3 regulates B cell function, we isolated B cells from the spleen of WT and Mettl3 cKO mice and incubated them with LPS (2 μg/ml), the ligand for CD40 (CD40L, 100 ng/ml), anti-IgM (1 μg/ml), or TNF-α (50 ng/ml) for 2 days. B cell activation was assessed by flow cytometry based on the expression level of CD69, CD86, and CD95. CD86 and CD95 showed that B cells from WT and Mettl3 cKO mice were activated at the same degree upon LPS, CD40L, and anti-IgM stimulation (Figures 4(a)-4(d) and Supplementary Figure 1D). However, the expression of CD69 showed a different pattern. B cells from Mettl3 cKO mice expressed higher activation marker CD69 in response to CD40L, anti-IgM, or TNF-α, while decreased in response to LPS (Figures 4(e)-4(h)). Besides, TNF-α also induced a higher CD95 expression level in Mettl3 cKO B cells (Figure 4(h)). However, IgG levels in the culture medium were comparable between WT and Mettl3 KO B cells upon LPS, CD40L, and anti-IgM stimulation (Figure 4(i)). The RT-qPCR analysis also showed consistent mRNA level of B cell survival factor Tnfsf13b (encoding BAFF), cytokines (Ltβ, Il10, and Tgfβ1), and chemokines (Cxcl12, Cxcl13, and Ccl21) in LPS-activated B cells from WT and Mettl3 cKO mice (Figure 4(j)). Next, we monitored the effects of Mettl3 knockout on B cell apoptosis under different treatments (Figure 5(a)). The proportion of early apoptotic cells (Annexin + PI -) was increased in Mettl3cKO B cells upon LPS and CD40L stimulation, while the late apoptotic cells (Annexin + PI + ) were more common in Mettl3cKO B cells with different stimuli (Figures 5(b)-5(e)). In addition, we analyzed B cell proliferation and observed that there was comparable residual CFSE fluorescence       Journal of Immunology Research apoptosis, and the effects on B cell activation varies from different stimuli.

Mettl3
Is Dispensable for the Profibrogenic Activity of B Cells in Liver Fibrosis. The above results showed that deletion of Mettl3 in B cells does not affect B cell specification but seems comprehensively affect B cell activation in vitro. B cell activation and function in vivo result from the integration of multiple signals and are much more complicated than in vitro. B cells can promote hepatic fibrosis progression [47,52]. However, the mechanisms that regulate B cell activation and function during liver fibrosis were not fully understood. Given the published dataset, we found that Mettl3 was upregulated in B cells of fibrotic livers [47] ( Figure 6(a)), indicating that Mettl3 was involved in B cell function in liver fibrosis. To identify whether Mettl3 regulates B cell function in vivo, we subjected WT and Mettl3 cKO mice to CCl 4 -induced liver fibrosis (Figure 6(b)), a well-established and widely-used hepatotoxic fibrosis model [53]. CCl 4 -induced liver fibrosis could reproduce pathological features of chronic liver diseases caused by various etiologies. This model also avoids activation of a specific subset of lymphocytes that occurred in LPS or Concanavalin Ainduced liver damage [54,55]. 24 hours after the last CCl 4 injection, flow cytometry assay for B cell activation in the spleen and liver was conducted. The results showed that B cell activation in Mettl3 cKO mice was the same as in WT controls, both in the spleen and liver (Figures 6(c) and 6(d)). Serum indicators of liver function showed no discernible difference between WT and Mettl3 cKO mice (Figure 6(e)). Liver fibrosis between WT and Mettl3 cKO mice was also comparable, evidenced by RT-qPCR ( Figure 6(f)) and western blot (Figure 6(g)) for profibrotic markers (Acta2 (encoding α-smooth muscle actin (αSMA)), Col1a1 (encoding collagen type I), and Pdgfrb (encoding Pdgfrβ)), H&E staining, PSR staining, and immunohistochemical staining for αSMA of mouse liver tissues (Figures 6(h)-6(j)). These results indicated that knocking out Mettl3 in B cells using Cd19-Cre does not affect the profibrogenic activity of B cells in liver fibrosis.

Discussion
Over the past decades, numerous studies have addressed that epigenetic modifications control various aspects of B cell development [56]. For instance, deficiency in BMI1 or MEL18 leads to a block of B cell development [57,58]. EZH2 or MYSM1 orchestrates early B cell development [59,60]. Loss of HADC1, HADC2, or HADC7 also impairs early B cell development [61,62]. m 6 A is the most frequent chemical modification of mRNA and lncRNA in eukaryotes. It controls multiple physiological and pathophysiological processes [14,15]. During B cell development, inhibition of Mettl3-mediated m 6 A modification in HSCs resulted in a block of HSC differentiation and subsequent decreased B cell frequency in the peripheral [30,31]. Deleting Mettl3 or Mettl14 in the very early stage of B cell specification using Mb1-Cre resulted in blockage of B cell differentiation in general, particularly pro-B to large pre-B and large pre-B to small pre-B transitions [41,42]. Since Cd19-Cre + mice express Cre from the pro-B cell stage on [44,45], later than Mb1-Cre + mice, we generated Cd19-Cre-mediated Mettl3 cKO mice to investigate the effect of m 6 A modification at later stages of B cell development and function. We observed that the ratio of total B cells in the Mettl3 cKO mice's peripheral blood, peritoneal cavity, and liver was equivalent to that in the WT controls. However, there is a slight increase in splenic B cell proportion in Mettl3 cKO mice. The percentage of pre-pro-B, pro-B, pre-B, immature, and mature B cells in the bone

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Journal of Immunology Research marrow of WT and Mettl3 cKO mice is also comparable, indicating that loss of Mettl3 from the pro-B stage on minimally affects B cell development. These results suggested that the requirement of Mettl3-mediated m 6 A during differentiation is stage-specific, and earlier stage deficiency may bring more severe outcomes.
B cells are an important part of the adaptive immune system by presenting antigens, secreting cytokines, and producing antibodies. B cells were mainly activated in T celldependent and T cell-independent manners in vivo [63,64]. For in vitro experiments, various stimulants, including LPS, sCD40L, anti-IgM, TNF-a, IFN-γ, and CpG ODNs, were used to trigger B cell activation. CD40L/CD40 ligandreceptor pair provides signals to B cells and induces T celldependent proliferation, survival, immunoglobulin class switching, antibody secretion, and germinal center formation [65]. LPS is also a potent stimulant of B cells and induces B cells to proliferate, produce antibodies, and secrete IL-6 through TLR4 [66][67][68]. Crosslinking the BCR with anti-IgM as a surrogate antigen induces T cell-independent B cell activation and moderate B cell proliferation [69,70]. TNF-α has been reported to be related to B cell proliferation, apoptosis, and the expression of individual molecules on the membrane, including CD19 and CD45 [71][72][73]. Thus, we isolated splenic B cells and treated them with LPS, CD40L, anti-IgM, or TNF-α to investigate whether depletion of Mettl3 in B cells could affect B cell activation, proliferation, and apoptosis in vitro. Our results indicated that loss of Mettl3 resulted in increased apoptosis with no difference in the proliferation and IgG production upon different 14 Journal of Immunology Research treatments. Cd19-Cre-mediated Mettl3 deletion makes B cells prone to apoptosis upon stimulation. However, different stimuli had different effects on B cell activation marker expression in vitro, consistent with previous studies that different stimulations may activate different subpopulations of B cells and result in different B cell phenotypes [74][75][76].
To see the integrated effect of different stimulation on Mettl3 cKO B cells, we used the CCl 4 -induced liver fibrosis model, in which Mettl3 in B cells was upregulated. We observed that Cd19-Cre-mediated Mettl3 deletion does not affect the profibrogenic activity of B cells in CCl 4 -induced liver fibrosis in vivo. B cells are traditionally known for producing antibodies and mediating humoral immune responses. However, recent studies showed that B cells are critical modulators of adaptive and innate immune responses [8]. Liver fibrosis is a dynamic wound-healing process characterized by the accumulation of extracellular matrix [77]. The role of B cells in liver fibrosis has been extensively explored recently [47,52]. Increased B cells in the fibrotic liver can exacerbate liver fibrosis in an antibody-independent manner by producing proinflammatory mediators to stimulate the hepatic stellate cells, a key driver of liver fibrosis [46]. Activated hepatic stellate cells produce retinoic acid, inducing B cell survival, plasma cell maturation, and IgG secretion [47]. The profibrotic role of B cells in the CCl 4 -induced liver fibrosis model depends on the myeloid differentiation primary response 88 (MYD88), which is indispensable for proper activation and proinflammatory cytokine production [47]. Here, we showed that the profibrogenic activity of B cells in liver fibrosis is independent of cell-autonomous Mettl3-mediated m 6 A modification.
During the preparation of this manuscript, another study that created Mettl3 flox/flox Cd19-Cre mice was published and showed consistent results with our research: no obvious developmental defects of B cells in Mettl3 flox/flox Cd19-Cre mice were observed. However, they found that Mettl3mediated m 6 A is essential for B cell survival and proliferation in the germinal center [43]. Furthermore, another study showed that m 6 A is vital for class switch recombination during the maturation of B cells [42]. In addition to our observation that B cell activation and apoptosis from Mettl3 knockout mice were differentially affected by different stimulants treatment in vitro, further exploration of Mettl3 on other aspects of B cell immunity with different models was worth further investigation.

Conclusion
Our work showed that Mettl3-mediated m 6 A is not required for B cell development, proliferation, and the profibrogenic function of B cells in liver fibrosis when deleted from the pro-B stage on using Cd19-Cre, strengthening the idea that B cell development and function are delicately controlled at different stages and contexts.

Data Availability
The experiment data used to support the findings of this study are included in the article.