The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud’s phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer’s disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects.


Introduction
Systemic sclerosis (SSc) is an autoimmune disease of unknown cause [1][2][3], a chronic disease characterized by vascular and immune dysfunction, and fibrosis of the skin and internal organs caused by excessive collagen deposition by activated fibroblasts [4][5][6][7].Pulmonary fibrosis and pulmonary arterial hypertension contribute to high mortality [8].The pathogenesis of SSc is very complex, involving both innate and adaptive immune responses in its development and progression [2,9].Autoimmune diseases are chronic, refractory diseases stimulated primarily by immune, hormonal, environmental, and genetic factors [10].Most autoimmune diseases are chronic diseases that are long-lasting or in some cases follow patients throughout their lives, and specific treatment methods have not yet been established [11].Although the mechanisms that cause clinical manifestations in SSc are also unknown [12], reactive oxygen species (ROS) are well known to be involved in the etiology of SSc [13][14][15][16][17][18][19][20].Raynaud's phenomenon (RP) occurs in 90% of SSc patients.After RP, patients may present with severe ischemia and ulceration, resulting in increased morbidity and mortality and decreased quality of life [21,22].RP causes frequent episodes of hypoxia-reperfusion [23], resulting in a positive feedback effect of luminal narrowing and ischemia, promoting the generation of ROS and free radicals.ROS can modulate cell activation and proliferation, and fibroblasts and endothelial cells are selectively targeted in SSc.Such an oxidative environment further exacerbates the disease by triggering endothelial damage, intimal thickening, and fibrosis.Ischemia and reperfusion induce oxidative stress (OS) [24,25] and inactivation of antioxidant enzymes [26].In addition, skin fibroblasts in SSc patients have been shown to elevate ROS [27], which triggers collagen synthesis [15,28], emphasizing the significant role of OS in SSc.
Twendee X ® (TwX) is an antioxidant combination comprising eight active ingredients: vitamin C, L-glutamine, niacin, L-cystine, coenzyme Q10, vitamin B2, succinic acid, and fumaric acid [29].Despite being a dietary supplement, it has undergone and passed stringent safety tests required for pharmaceuticals including chromosomal aberration test, toxicity test, and mutation test.
TwX has demonstrated various beneficial effects including protecting mitochondria, increasing ATP production, reducing blood OS, maintaining autophagy and neurogenesis, and elongating telomeres [30].The effect of TwX on cognitive function has also been studied in varying disease models.A multicenter randomized, double-blind, placebocontrolled intervention clinical trial has demonstrated its potential to prevent dementia in Japanese patients with mild cognitive impairment (MCI) [31].Moreover, TwX has shown positive outcomes in mouse models of Alzheimer's disease (AD) with chronic cerebral hypoperfusion (CCH + APP23 mice); motor coordination and working memory were improved and hippocampal neurons were restored.Other outcomes derived from TwX include significant improvement of cognitive impairment, reduction of Aβ pathology and neuronal loss, and alleviation of neural inflammation and OS [32,33].
In a mouse model of ischemic stroke, pretreatment with TwX (20 mg/kg/d) for 14 days not only reduced infarct size but also decreased the expression of OS markers and tumor necrosis factor-α (TNF-α) and inflammation markers [34].Recent reports have also suggested the potential of TwX to enhance the quality of life in dementia patients by influencing gut microbiota [35].The effects of TwX shown in previous studies could be effective enough to control OS in SSc.The aim of this study was to investigate the effects of TwX on various human-like SSc symptoms [36,37] in mice that were injected with HOCl, an oxidation accelerator, into their dermis [36,[38][39][40][41][42].
Serum AOPP concentrations in SSc patients are usually reported to be higher than in healthy subjects [13,43,44].This was also observed in the present study; HOCl-induced SSc mice had higher AOPP than PBS-treated mice.AOPP is a marker of OS.Serum AOPP is involved in the production of ROS and regulates fibroblast proliferation in SSc patients [13].HOCl-induced AOPP (HOCl-AOPP) induces H 2 O 2 rather than NO and activates both endothelial cells and fibroblasts, and a dose-dependent proliferative response can be observed [13].In fact, serum from SSc patients with RP shows significantly higher levels of H 2 O 2 and proliferation of NIH3T3 fibroblasts compared to serum from SSc patients without RP and healthy subjects [13].Additionally, decreased concentrations of antioxidants such as ascorbic acid, α-tocopherol, and β-carotene, as well as lower selenium levels have been reported in SSc patients [26].This increase in ROS and deficiency in antioxidant capacity increases OS and contributes to the development of free-radical-mediated damage.This elevated OS is further exacerbated not only by inflammatory processes but also by frequent reperfusion injury such as that seen in RP [45].Vascular changes including microvascular endothelial injury, vasospasm tendency with an inadequate vasodilatory response, changes in the coagulation/fibrinolytic system, and proliferation of intimal cells leading to microvascular system occlusion are significant events in the early stages of SSc, and it highlights the crucial role of OS in the early stages of SSc [46] and in the progression and worsening of the disease.Serum AOPP concentrations in SSc patients are usually reported to be highe healthy subjects [13,43,44].This was also observed in the present study; HOCl SSc mice had higher AOPP than PBS-treated mice.AOPP is a marker of OS.Seru is involved in the production of ROS and regulates fibroblast proliferation in SSc [13].HOCl-induced AOPP (HOCl-AOPP) induces H2O2 rather than NO and activ endothelial cells and fibroblasts, and a dose-dependent proliferative response ca served [13].In fact, serum from SSc patients with RP shows significantly higher H2O2 and proliferation of NIH3T3 fibroblasts compared to serum from SSc patie out RP and healthy subjects [13].Additionally, decreased concentrations of anti such as ascorbic acid, α-tocopherol, and β-carotene, as well as lower selenium lev been reported in SSc patients [26].This increase in ROS and deficiency in antiox pacity increases OS and contributes to the development of free-radical-mediated This elevated OS is further exacerbated not only by inflammatory processes bu frequent reperfusion injury such as that seen in RP [45].Vascular changes inclu crovascular endothelial injury, vasospasm tendency with an inadequate vasodil sponse, changes in the coagulation/fibrinolytic system, and proliferation of inti leading to microvascular system occlusion are significant events in the early stage and it highlights the crucial role of OS in the early stages of SSc [46] and in the pro and worsening of the disease.
Based on the above, it is quite possible that reducing AOPP may suppress H reducing OS at some point is one pathway to alleviate the various symptoms of S Based on the above, it is quite possible that reducing AOPP may suppress H 2 O 2 , and reducing OS at some point is one pathway to alleviate the various symptoms of SSc.TwX has been shown to protect cells and mitochondria by lowering cellular and mitochondrial ROS levels and increasing Mn-SOD and Cu/Zn-SOD activity [30].These effects significantly suppress OS caused by blood hydro-peroxides and other substances in CCH + APP23 mice and in OPP rats treated with orthophenyl phenol (OPP), a fruit and another preservative that induces ROS in the body [32,33,35].In the present study, TwX affected OS to significantly reduce HOCl-derived oxidative damage.
TwX (20 mg/kg/d) significantly suppressed OH proline levels (−32% (p <0.05) for the skin -64% (p < 0.001) for the lung) and Col1 mRNA expression (−20% (p < 0.01)), thereby suppressing collagen accumulation (Figure 2B-D).Further observations, confirmed by histopathological images, showed that skin thickness induced by HOCl was gradually suppressed from day 14 onward and significantly reduced to a thickness similar to that of PBS + TwX mice from day 20 onward (−25% on day 35 (p < 0.05)) (Figure 2A,E).Similarly, the lungs of HOCl-induced SSc mice showed high concentrations of OH proline (+36% (Not significant)) (Figure 2C), and TwX significantly reduced this collagen accumulation to lower levels than in PBS control mice (-64%, p < 0.001).Several human skin pathologies, including SSc, are associated with significant redox imbalance at the cellular level [13,[47][48][49].Antioxidant administration has also been reported to suppress skin and pulmonary fibrosis in SSc [50][51][52].One example is Edaravone a novel, free radical scavenger and neuroprotective agent used in the treatment of acute Several human skin pathologies, including SSc, are associated with significant redox imbalance at the cellular level [13,[47][48][49].Antioxidant administration has also been reported to suppress skin and pulmonary fibrosis in SSc [50][51][52].One example is Edaravone, a novel, free radical scavenger and neuroprotective agent used in the treatment of acute embolic stroke in humans [53].The skin fibrosis score in bleomycin-induced mouse models (BLM mice) increases over time, and while Edaravone significantly reduced dermal skin thickness, it did not lower to the level of the control mice [50].TwX also had an effect on skin and lung fibrosis in HOCl-induced SSc mice.Although the effect of TwX on dermal thickening was not observed in the early stages of HOCl treatment, the effect emerged gradually and eventually decreased dermal thickness to a state close to that of the PBS control mice.
Although TwX also has free radical scavenging [54] and neuroprotective effects [30], it may be necessary to use more immediate agents to improve skin thickening from the initial stage.OS scavenging could lead to a rational targeted therapeutic approach for SSc; however, the complexity and repetitive nature of the cascading system in SSc make it impossible to achieve efficacy with a treatment derived from a single antioxidant [26].The composition of TwX, which includes eight high-efficacy antioxidants, may underlie its effectiveness in alleviating SSc symptoms to a state similar to the PBS control mice.The same could be true for collagen content in skin and lungs.
The expression of α-SMA protein was significantly enhanced in HOCl-induced SSc mice (+142% (p < 0.05)) (Figure 3A).However, TwX significantly reduced it to a level similar to that of PBS control mice (−50% (p < 0.05)) (Figure 3A).thickening was not observed in the early stages of HOCl treatment, the effect emerged gradually and eventually decreased dermal thickness to a state close to that of the PBS control mice.
Although TwX also has free radical scavenging [54] and neuroprotective effects [30], it may be necessary to use more immediate agents to improve skin thickening from the initial stage.OS scavenging could lead to a rational targeted therapeutic approach for SSc; however, the complexity and repetitive nature of the cascading system in SSc make it impossible to achieve efficacy with a treatment derived from a single antioxidant [26].The composition of TwX, which includes eight high-efficacy antioxidants, may underlie its effectiveness in alleviating SSc symptoms to a state similar to the PBS control mice.The same could be true for collagen content in skin and lungs.
α-SMA is expressed primarily in vascular smooth muscle and is involved in the differentiation of fibroblasts into myofibroblasts, which are responsible for the production of extracellular matrix in fibrotic diseases such as SSc.It has been reported that the development of ROS in SSc fibroblasts increases the expression of type 1 collagen and α-SMA genes [55], while the expression of type I collagen and α-SMA genes also activates ROS [15,28].In other words, a vicious cycle between the pathology and ROS is indicated.TwX suppressed fibrosis in SSc by decreasing α-SMA as well as ROS.This suggests that TwX is promising as an antioxidant compound to inhibit fibrosis by breaking the vicious cycle between pathology and ROS.In contrast, the expression of H-Ras protein was significantly reduced in HOCl-induced SSc mice (−51% (p < 0.05)) (Figure 3B).H-Ras proteins are primarily involved in the regulation of cell division.When the protein binds to GDP, it does not relay signals to the cell's nucleus to divide.H-Ras-GTPase proteins are activated for example during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-β-induced α-SMA is expressed primarily in vascular smooth muscle and is involved in the differentiation of fibroblasts into myofibroblasts, which are responsible for the production of extracellular matrix in fibrotic diseases such as SSc.It has been reported that the development of ROS in SSc fibroblasts increases the expression of type 1 collagen and α-SMA genes [55], while the expression of type I collagen and α-SMA genes also activates ROS [15,28].In other words, a vicious cycle between the pathology and ROS is indicated.TwX suppressed fibrosis in SSc by decreasing α-SMA as well as ROS.This suggests that TwX is promising as an antioxidant compound to inhibit fibrosis by breaking the vicious cycle between pathology and ROS.
In contrast, the expression of H-Ras protein was significantly reduced in HOCl-induced SSc mice (−51% (p < 0.05)) (Figure 3B).H-Ras proteins are primarily involved in the regulation of cell division.When the protein binds to GDP, it does not relay signals to the cell's nucleus to divide.H-Ras-GTPase proteins are activated for example during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-β-induced epithelial-mesenchymal transition [56].The results suggest that normal homeostatic functions may act to protect cells from HOCl-induced excessive division, resulting in a decrease in H-Ras.On the other hand, this decrease was not observed in PBS control or HOCl + TwX mice, likely due to the absence of induced fibrosis.We speculate that TwX prevented fibrosis before the normal homeostatic function was activated in H-Ras.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 14 epithelial-mesenchymal transition [56].The results suggest that normal homeostatic functions may act to protect cells from HOCl-induced excessive division, resulting in a decrease in H-Ras.On the other hand, this decrease was not observed in PBS control or HOCl + TwX mice, likely due to the absence of induced fibrosis.We speculate that TwX prevented fibrosis before the normal homeostatic function was activated in H-Ras.
Macrophages are inflammatory cells that produce other immune mediators and cytokines with both protective and pro-inflammatory functions [58].In SSc, macrophages also play an important role in the disease pathogenesis.When macrophages are activated, they release mediators and express surface markers, and normally the activation of both alternative macrophages (M2) and inflammatory macrophages (M1) is associated with SSc [59].Macrophages are the predominant immune cell population in SSc pathology, and their dysfunction leads to abnormal repair and regeneration with runaway inflammatory mediators and growth factors [60].Excessive accumulation of M2 macrophages is closely associated with fibrosis [61,62], which results from the abnormal accumulation of extracellular matrix (ECM) components such as collagen and fibronectin.ECM promotes wound healing and tissue repair in mild tissue injury; however, in severe injury, excessive accumulation of ECM can disrupt tissue structure and lead to organ dysfunction [63].Thus, macrophages play a crucial role in fibrosis pathogenesis [64,65], especially M2a macrophages, which significantly promote fibrosis progression [62,66].The present study suggests that a shift in macrophage balance toward the M2 phenotype occurred during the chronic phase (after the 21st day of the experiment) in HOCl-induced SSc mice.However, the trend was reversed when TwX was administered, resulting in the accumulation of M1 macrophages over M2 macrophages, thereby slowing the fibrotic process.Macrophages are inflammatory cells that produce other immune mediators and cytokines with both protective and pro-inflammatory functions [58].In SSc, macrophages also play an important role in the disease pathogenesis.When macrophages are activated, they release mediators and express surface markers, and normally the activation of both alternative macrophages (M2) and inflammatory macrophages (M1) is associated with SSc [59].Macrophages are the predominant immune cell population in SSc pathology, and their dysfunction leads to abnormal repair and regeneration with runaway inflammatory mediators and growth factors [60].Excessive accumulation of M2 macrophages is closely associated with fibrosis [61,62], which results from the abnormal accumulation of extracellular matrix (ECM) components such as collagen and fibronectin.ECM promotes wound healing and tissue repair in mild tissue injury; however, in severe injury, excessive accumulation of ECM can disrupt tissue structure and lead to organ dysfunction [63].Thus, macrophages play a crucial role in fibrosis pathogenesis [64,65], especially M2a macrophages, which significantly promote fibrosis progression [62,66].The present study suggests that a shift in macrophage balance toward the M2 phenotype occurred during the chronic phase (after the 21st day of the experiment) in HOCl-induced SSc mice.However, the trend was reversed when TwX was administered, resulting in the accumulation of M1 macrophages over M2 macrophages, thereby slowing the fibrotic process.
Subcutaneous injections of 200 µL of HOCl into the back of the mice were administered once daily for 6 weeks, as previously described [38,39].The PBS control group and PBS + TwX group received injections of 100 µL of sterilized PBS.
One month before and during the 6 weeks of HOCl injections, the mice consumed plain tap water or water with TwX ad libitum (20 mg/kg/d).Two days after the final injection, the animals were euthanized by cervical dislocation, and samples of sera, spleen, lungs, and skin biopsies were collected.Tissue samples were fixed in 10% acetic acid formol for histopathological analysis.

Evaluating Fibrosis
Fibrosis of the skin was assessed weekly in vivo, by measuring the dermal thickness of the shaved backs of the mice.This assessment was conducted under double-blinded conditions using a caliper and expressed in millimeters once a week until the end of the experiment.

Serum AOPP Measurement
Serum AOPP concentration was measured by spectrophotometry as previously described [44].Assays of AOPP in sera were diluted (1:5) in PBS and distributed (200 µL) onto a 96-well plate with 10 µL of 1.16 M potassium iodide.Calibration used a twofold dilution series of chloramine-T solution within a range of 0 to 100 mM.The absorbance was read at 340 nm on a microplate reader (Fusion; PerkinElmer, Wellesley, MA, USA), and AOPP concentration was expressed as mM of chloramine-T equivalents.

Collagen Content Measurement
The collagen content was assessed in the skin and lungs using the OH-proline content evaluation as recommended by Woessner [67].Briefly, after completing punch biopsies (5 mm diameter), the samples were incubated in HCl (6 M) for 3 h at 120 • C. The pH of the samples was adjusted to 7 and then mixed with chloramine T (0.06 M) and incubated for 20 min at room temperature.Perchloric acid (3.15 M) and p-dimethylaminobenzaldehyde (20%) were then added, and samples were incubated for an additional 20 min at 60 • C. The absorbance was determined at 557 nm with a Fusion microplate spectrophotometer (PerkinElmer, Wellesley, MA, USA).

Histopathologic Analysis
A 5 µm thick tissue section was prepared from the mid-portion of paraffin-embedded lung and skin sample and stained with hematoxylin and eosin.Slides were examined by standard brightfield microscopy (Olympus BX60, Tokyo, Japan) by a pathologist who was blinded to the animal's group assignment.

Evaluation of mRNA Expressions
The qRT-PCR technique was conducted as follows: the skin samples taken from the four groups of mice were immediately frozen in liquid nitrogen.Total RNA was extracted from the mouse tissue using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions.

Evaluating Immune Status
Spleen cell suspensions were prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium.For each mouse, splenocytes were quantified using a Malassez counting chamber.Cells were then incubated with an antibody at 4 • C for 30 min in the dark in PBS with 2% normal FBS.Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to standard techniques.To characterize splenic cells, the monoclonal antibodies used spleen cell suspensions prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium.For each mouse, splenocytes were quantified using a Malassez counting chamber.Cells' suspensions were incubated for 20 min with the following antibodies.All antibodies were obtained from Biolegend (San Diego, CA, USA) and used at a dilution of 1:100 unless otherwise mentioned: CD3 FITC, CD4 APC Fire 750 (BD Biosciences, San Jose, CA, USA), CD8 BV 605, CD69 PEDazzle594, CD40 PercPCy5.5,B220 APC, CD44 BV650, MHC II eFluor450 (DAPI) (eBioscience, San Diego, CA, USA), F4/80 BV711 (dilution 1:200), CD11b PercP Cy5.5, CD80 BV421, CD86 FITC, CD206 Alexa Fluor 647 (BD Biosciences, San Jose, CA, USA.Dilution 1:200), Ly6C PECy7.

Statistical Analysis
All the results are presented as the means ± SDs.Kruskal-Wallis test with Dunn's multiple comparison test was used for all statistical analysis.A p value less than 0.05 was considered statistically significant.

Conclusions
The HOCl-induced SSc model mice used in this study were artificially reproduced, and not all of them are applicable to human SSc symptoms.However, among the SSc induction models using different reagents, the HOCl-induced mouse model used in this study is the model that best reproduces human SSc symptoms.The present study suggests that TwX may delay the transition to the chronic phase in the HOCl-induced SSc mouse model by reducing skin thickening and suppressing skin and lung fibrosis.This indicates that TwX can be expected to alleviate symptoms against SSc in humans.Furthermore,

Figure 1 .
Figure 1.Effect of Twendee X ® on sera redox status.Concentrations of advanced oxidatio products (AOPP) in the sera from mice (mM of chloramine T equivalent).Each box represe ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison used for statistical analysis).* p < 0.05.

Figure 1 .
Figure 1.Effect of Twendee X ® on sera redox status.Concentrations of advanced oxidation protein products (AOPP) in the sera from mice (mM of chloramine T equivalent).Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison test was used for statistical analysis).* p < 0.05.

Figure 3 .
Figure 3.Effect of Twendee X ® on fibroblast differentiation.(A) α-SMA and (B) H-Ras in skin were evaluated by Western blot.Results were normalized to tubulin.Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison test was used for all statistical analysis.* p < 0.05.

Figure 3 .
Figure 3.Effect of Twendee X ® on fibroblast differentiation.(A) α-SMA and (B) H-Ras in skin were evaluated by Western blot.Results were normalized to tubulin.Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison test was used for all statistical analysis.* p < 0.05.

Figure 4 .
Figure 4. Effect of Twendee X ® on Collagen and cytokines expression.Relative mRNA level of (A) Il-6, (B) Il-33, and (C) Il-17 mRNA levels in skin evaluated by qRT-PCR.Results were normalized to GAPDH.Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison test was used for all statistical analysis.No significant difference was observed.

Figure 4 .
Figure 4. Effect of Twendee X ® on Collagen and cytokines expression.Relative mRNA level of (A) Il-6, (B) Il-33, and (C) Il-17 mRNA levels in skin evaluated by qRT-PCR.Results were normalized to GAPDH.Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison test was used for all statistical analysis.No significant difference was observed.

Figure 5 .
Figure 5. Effects of Twendee X ® on B and T CD4+ cells activation assessed by flow cytometry in SS mice.The side-scatter (SSC) and the forward-scatter channels (FSC) were used to gate the leukocyte A total of 100,000 events were accumulated for each sample.Doublets were excluded with FSC-A and FSC-H channels.(A,B) Activation of B220+ cells assessed by CD40 expression (A) and MHC expression (B).(C,D) Activation of CD4+ T cells assessed by CD69 (C) and CD44 (D) expression Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's mu tiple comparison test was used for all statistical analysis.* p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5 .
Figure 5. Effects of Twendee X ® on B and T CD4+ cells activation assessed by flow cytometry in SSc mice.The side-scatter (SSC) and the forward-scatter channels (FSC) were used to gate the leukocytes.A total of 100,000 events were accumulated for each sample.Doublets were excluded with FSC-A and FSC-H channels.(A,B) Activation of B220+ cells assessed by CD40 expression (A) and MHC II expression (B).(C,D) Activation of CD4+ T cells assessed by CD69 (C) and CD44 (D) expression.Each box represents mean ± SD from n = 10 individual mice.Kruskal-Wallis test with Dunn's multiple comparison test was used for all statistical analysis.* p < 0.05; ** p < 0.01; *** p < 0.001.