Chemotherapy-induced microbiota exacerbates the toxicity of chemotherapy through the suppression of interleukin-10 from macrophages
化疗诱导的微生物群通过抑制巨噬细胞分泌的白介素-10 加剧化疗毒性

Alteration of gut microbiota mediates chemotherapy-induced toxicity
肠道微生物群的改变介导化疗诱导的毒性

To induce chemotherapy-induced toxicity, we administered a high dose of oxaliplatin (20 mg/kg) to specific pathogen-free (SPF) C57BL/6 mice every five days (Figure 1(a)). Mice exposed to high-dose oxaliplatin exhibited heighten weight loss¹⁰ and worse clinical scores¹⁵ (e.g., weight loss, hunched posture, ruffled hair coat, reluctance to move, and other performance) than those in the control group (Figures 1(b–c)). Only half of oxaliplatin-exposed mice exhibited long-term survival (20 days) (Figure 1(d)). Routine blood parameters showed that the levels of red and white blood cells, platelets, and hemoglobin significantly decreased after high-dose chemotherapy (Figure 1(e)). Moreover, oxaliplatin-exposed mice showed significantly decreased splenic white and red pulp regions (Figure 1(f)), indicating impairment of the hematopoietic system. Intestinal histological assessment showed that the gaps between the crypt bases and muscularis mucosa were significantly larger in oxaliplatin-exposed mice than in the control group (Figure 1(g)).
为了诱导化疗引起的毒性，我们每五天向特定病原体无菌（SPF）C57BL/6 小鼠给予高剂量奥沙利铂（20 mg/kg）（ Figure 1(a) ）。接受高剂量奥沙利铂的小鼠表现出更明显的体重减轻 ¹⁰ 和更差的临床评分 ¹⁵ （如体重减轻、驼背、毛发蓬乱、不愿活动及其他表现），相比对照组（ Figures 1(b–c) ）。仅有一半接受奥沙利铂的小鼠表现出长期存活（20 天）（ Figure 1(d) ）。常规血液参数显示，高剂量化疗后红细胞、白细胞、血小板和血红蛋白水平显著下降（ Figure 1(e) ）。此外，奥沙利铂处理的小鼠脾脏白髓和红髓区域显著减少（ Figure 1(f) ），提示造血系统受损。肠道组织学评估显示，奥沙利铂处理组小鼠隐窝基底与黏膜肌层之间的间隙显著大于对照组（ Figure 1(g) ）。

To further describe the chemotherapy-induced toxicity, a tumor-bearing mice model was established by subcutaneous injection of MC38 CRC cells. High dose of oxaliplatin (20 mg/kg) was subsequently injected into mice as shown in Figure 1(h). Similar to the results described above, mice exposed to the high dose of oxaliplatin exhibited an exacerbated weight loss and a worse clinical score, as well as a half of long term survival (Figures 1(i–k)). Similar change of routine blood parameters also validated the toxicity induced by chemotherapy (Figure 1(l)). Meanwhile, decreased splenic pulp regions and increased gap between the crypt bases and muscularis mucosa were found in oxaliplatin-exposed mice (Figure 1(m,n)). In addition to the toxicity associated parameters, significant alleviation of tumor growth and a corresponding reduction in tumor size and weight were observed in mice with high dose of oxaliplatin (Figures 1(o–q)). These two mice models indicate that mice exposed to high-dose oxaliplatin exhibit severe systemic side effects and toxicity to the hematopoietic and gastrointestinal systems, simulating toxicity in patients suffering from chemotherapeutics.
为了进一步描述化疗引起的毒性，建立了肿瘤负荷小鼠模型，通过皮下注射 MC38 结直肠癌细胞。随后如 Figure 1(h) 所示，向小鼠注射高剂量奥沙利铂（20 mg/kg）。与上述结果类似，接受高剂量奥沙利铂的小鼠表现出加重的体重减轻和更差的临床评分，以及长期生存率减半（ Figures 1(i–k) ）。常规血液参数的类似变化也验证了化疗引起的毒性（ Figure 1(l) ）。同时，在奥沙利铂暴露的小鼠中发现脾髓区减少，隐窝基底与黏膜肌层之间的间隙增大（ Figure 1(m,n) ）。除了与毒性相关的参数外，高剂量奥沙利铂小鼠还表现出肿瘤生长显著缓解，肿瘤大小和重量相应减少（ Figures 1(o–q) ）。这两种小鼠模型表明，接受高剂量奥沙利铂的小鼠表现出严重的全身副作用及对造血和胃肠系统的毒性，模拟了接受化疗患者的毒性反应。

To explore whether gut microbiota has a causal effect on chemotherapy-induced toxicity, we collected feces from mice without subcutaneous tumors treated with high doses of oxaliplatin and performed fecal microbiota transplantation (FMT) in healthy C57BL/6 recipient mice. We also demonstrated that there was no oxaliplatin detected in feces from mice given multiple intraperitoneal injections of oxaliplatin by liquid chromatography-mass spectrometry (LC-MS) (Figure S1(a)). All recipient mice (OXA-FMT and control-FMT) were challenged with oxaliplatin (Figure 2(a)). Interestingly, OXA-FMT mice exhibited heightened weight loss, worse clinical scores, shorter survival durations, and worse routine blood parameters than the control-FMT group (Figures 2(b–e)). Histological analysis further showed a significant decrease in bone marrow cellularity and splenic loss of white and red pulp regions, as well as larger gaps between the crypt bases and muscularis mucosa in the OXA-FMT group than in the control-FMT group (Figures 2(f–h)). These findings demonstrate that the microbiota from oxaliplatin-exposed mice exacerbates chemotherapy-induced toxicity in recipient mice.
为了探究肠道微生物群是否对化疗诱导的毒性具有因果作用，我们收集了未植入皮下肿瘤且接受高剂量奥沙利铂治疗的小鼠粪便，并在健康的 C57BL/6 受体小鼠中进行了粪便微生物群移植（FMT）。我们还通过液相色谱-质谱联用技术（LC-MS）证明，在多次腹腔注射奥沙利铂的小鼠粪便中未检测到奥沙利铂（图 S1(a)）。所有受体小鼠（OXA-FMT 组和对照-FMT 组）均接受了奥沙利铂挑战（ Figure 2(a) ）。有趣的是，OXA-FMT 小鼠表现出更明显的体重减轻、更差的临床评分、更短的存活时间以及比对照-FMT 组更差的常规血液参数（ Figures 2(b–e ）。组织学分析进一步显示，OXA-FMT 组骨髓细胞密度显著降低，脾脏白髓和红髓区域丧失，隐窝基底与黏膜肌层之间的间隙较对照-FMT 组更大（ Figures 2(f–h ）。这些发现表明，来自奥沙利铂暴露小鼠的微生物群加剧了受体小鼠的化疗诱导毒性。

Microbiota-mediated toxicity of chemotherapy is macrophage-dependent
化疗相关的微生物群介导的毒性依赖于巨噬细胞

To clarify the mechanisms by which the microbiota mediates chemotherapy-induced toxicity, we performed transcriptional analysis of splenic cells obtained from OXA-FMT mice and control group. The splenic transcriptome of the OXA-FMT group was significantly different from that of the control-FMT group (Figure 3(a)). We then evaluated the relative abundance of different immune cells using the CIBERSORT algorithm and found that the fraction of monocytes and macrophages changed significantly (Figure 3(b)). An increased proportion of monocytes and decreased proportion of M2 macrophages were observed in the OXA-FMT group (Figure 3(b)). Alteration of several differential genes associated with monocytes and M2 macrophages was also observed after FMT intervention (Figure S1(b)). Furthermore, immunohistochemistry (IHC) indicated that the proportion of macrophages decreased, rather than the proportion of CD4⁺ T cells and Treg cells (Figures S1(c–h)). We further evaluated the differential genes referred in CIBERSORT algorithm. In addition to the involvement of different immune cell signaling pathways, we found that the differential genes were also clustered in the Toll-like receptor signaling pathway and NF-κB signaling pathway (Figure 3(c)).
为阐明微生物群介导化疗毒性的机制，我们对来自 OXA-FMT 小鼠和对照组的脾脏细胞进行了转录组分析。OXA-FMT 组的脾脏转录组与对照-FMT 组显著不同（ Figure 3(a) ）。随后，我们使用 CIBERSORT 算法评估了不同免疫细胞的相对丰度，发现单核细胞和巨噬细胞的比例发生了显著变化（ Figure 3(b) ）。OXA-FMT 组中单核细胞比例增加，而 M2 型巨噬细胞比例减少（ Figure 3(b) ）。FMT 干预后，与单核细胞和 M2 型巨噬细胞相关的多个差异基因也发生了变化（图 S1(b)）。此外，免疫组化（IHC）显示巨噬细胞比例下降，而 CD4 ⁺ T 细胞和调节性 T 细胞（Treg）比例未见下降（图 S1(c–h)）。我们进一步评估了 CIBERSORT 算法中提及的差异基因。 除了不同免疫细胞信号通路的参与外，我们还发现差异基因聚集在 Toll 样受体信号通路和 NF-κB 信号通路中（ Figure 3(c) ）。

To subsequent confirm the role of immune cell in chemotherapy-induced toxicity, we next applied FMT experiments with recombination activating gene 1 (Rag-1)-deficient (Rag1^-/-) mice lacking mature B and T lymphocytes (Figure 3(d)). Rag1^-/- mice gavaged with feces from oxaliplatin-treated donors exhibited greater body weight loss, higher clinical scores, and shorter survival durations (Figures 3(e–i)), implicating the other potential immune response such as innate immunity were involved in microbiota-mediated chemotherapy toxicity. While immunologic memory is a key feature of adaptive immunity, more recently the term “trained innate immunity” has been used to describe innate immune cells, primarily macrophages that exhibit enhanced responsiveness upon reinfection.¹⁶ To investigate the role of macrophages in chemotherapy-induced toxicity, recipient mice were intraperitoneally injected with clodronate to eliminate macrophages, followed by FMT, as described above (Figure 3(j)). Flow cytometric analysis confirmed that macrophages were depleted in splenic cells of mice treated with clodronate (Figure S1(i)). Mice with undepleted macrophages exhibited heightened chemotherapeutic-induced toxicity in the OXA-FMT group compared to the control-FMT group. Interestingly, similar weight loss, clinical score, and pathologic features were observed between OXA-FMT and control-FMT in macrophage-depleted recipient mice, which were both significantly lower than those in mice with intact macrophages (Figures 3(k–p)). Collectively, these findings demonstrate that microbiota-mediated chemotherapy-induced toxicity is dependent on macrophage function.
为了进一步确认免疫细胞在化疗诱导毒性中的作用，我们接着进行了重组激活基因 1（Rag-1）缺陷（Rag1^0）小鼠的粪菌移植（FMT）实验，这些小鼠缺乏成熟的 B 细胞和 T 细胞（ Figure 3(d) ）。用来自奥沙利铂处理供体的粪便灌胃的 Rag1^2 小鼠表现出更明显的体重减轻、更高的临床评分和更短的生存时间（ Figures 3(e–i ），这表明其他潜在的免疫反应如先天免疫也参与了微生物群介导的化疗毒性。虽然免疫记忆是适应性免疫的关键特征，但近年来“训练性先天免疫”一词被用来描述主要是巨噬细胞的先天免疫细胞在再次感染时表现出增强的反应性（ ¹⁶ ）。为探究巨噬细胞在化疗诱导毒性中的作用，受体小鼠腹腔注射氯膦酸盐以消除巨噬细胞，随后进行上述的 FMT（ Figure 3(j) ）。流式细胞术分析证实，氯膦酸盐处理的小鼠脾细胞中的巨噬细胞被有效清除（图 S1(i)）。 未去除巨噬细胞的小鼠在 OXA-FMT 组中表现出比对照-FMT 组更严重的化疗诱导毒性。有趣的是，在巨噬细胞耗竭的受体小鼠中，OXA-FMT 组和对照-FMT 组之间的体重减轻、临床评分和病理特征相似，且均显著低于巨噬细胞完整的小鼠（ Figures 3(k–p) ）。综上所述，这些发现表明，微生物群介导的化疗诱导毒性依赖于巨噬细胞功能。

Suppression of IL-10 is responsible for chemotherapy-induced toxicity
IL-10 的抑制是化疗诱导毒性的原因

To identify the most prominent immune response induced by the gut microbiota in chemotherapy-induced toxicity, we measured the expression of 31 serum cytokines in the OXA-FMT and control-FMT mice (Figure 4(a)). Mice colonized with oxaliplatin-treated microbiota exhibited different serum cytokine levels compared with control-FMT mice. Specifically, significant downregulation of IL-10 was observed in mice colonized with oxaliplatin-treated microbiota (Figure 4(a)). Moreover, IHC experiments confirmed that the expression of IL-10 in the colon and spleen of OXA-FMT group mice was significantly decreased compared to that in the control-FMT group (Figures 4(b–c)). IL-10 is an important cytokine that suppresses the inflammatory response. To explore the role of IL-10 in chemotherapy-induced toxicity, we intraperitoneally injected oxaliplatin into Il10^-/- and wild-type (WT) mice (Figure S2(a)). Interestingly, Il10^-/- mice exhibited worse weight loss and clinical scores, as well as the exacerbation of histological features (Figures S2(b–f)). To confirm the role of IL-10, we intraperitoneally injected recombinant IL-10 (rIL-10) into C57BL/6 mice (Figure 4(d)). Mice administered rIL-10 exhibited significantly lower weight loss and improved clinical scores upon oxaliplatin exposure (Figures 4(e–f)). Additionally, administration of rIL-10 also rescued the exacerbation of splenic white and red pulp regions as well as the gaps between the crypt bases and muscularis mucosa, indicating an improvement in hematopoietic and gastrointestinal toxicity (Figures 4(g–j)). Moreover, rIL-10 treatment increased the mRNA levels of epithelial tight junctions, such as ZO-1 and occludin in the colon, suggesting increased barrier function (Figure 4(k)). These data demonstrate that the microbiota-mediated downregulation of IL-10 expression is responsible for the exacerbation of chemotherapy-induced toxicity.
为了确定肠道微生物群在化疗诱导毒性中引发的最显著免疫反应，我们测量了 OXA-FMT 组和对照-FMT 组小鼠血清中 31 种细胞因子的表达（ Figure 4(a) ）。移植了奥沙利铂处理微生物群的小鼠，其血清细胞因子水平与对照-FMT 组小鼠存在差异。具体而言，移植了奥沙利铂处理微生物群的小鼠中 IL-10 显著下调（ Figure 4(a) ）。此外，免疫组化实验确认，OXA-FMT 组小鼠结肠和脾脏中 IL-10 的表达显著低于对照-FMT 组（ Figures 4(b–c) ）。IL-10 是一种重要的细胞因子，能够抑制炎症反应。为探究 IL-10 在化疗诱导毒性中的作用，我们向 Il10 ^-/- 和野生型（WT）小鼠腹腔注射奥沙利铂（图 S2(a)）。有趣的是，Il10 ^-/- 小鼠表现出更严重的体重减轻和临床评分恶化，以及组织学特征的加重（图 S2(b–f)）。为确认 IL-10 的作用，我们向 C57BL/6 小鼠腹腔注射重组 IL-10（rIL-10）（ Figure 4(d) ）。 给予小鼠重组 IL-10（rIL-10）后，在接受奥沙利铂治疗时，体重减轻显著减少，临床评分改善（ Figures 4(e–f) ）。此外，rIL-10 的给予还缓解了脾脏白髓和红髓区域的恶化，以及隐窝基底与黏膜肌层之间的间隙，表明造血和胃肠道毒性得到改善（ Figures 4(g–j ）。此外，rIL-10 治疗还提高了结肠中上皮紧密连接蛋白如 ZO-1 和闭合蛋白的 mRNA 水平，提示屏障功能增强（ Figure 4(k) ）。这些数据表明，微生物群介导的 IL-10 表达下调是化疗诱导毒性加重的原因。

Downregulation of IL-10 from macrophage mediates the chemotherapy-induced toxicity
巨噬细胞来源的 IL-10 下调介导化疗诱导的毒性

We next determined the source of IL-10 secretion by flow cytometry. Flow cytometry analysis of splenocytes showed that F4/80⁺ IL-10⁺ macrophages were markedly suppressed in recipient mice in the OXA-FMT group compared with those in the control-FMT group (Figure 5(a)). Interestingly, both CD4⁺ IL-10⁺ T cells and CD4⁺Foxp3⁺ IL-10⁺ regulatory T cells in splenocytes were similar between this two groups (Figure 5(a)). Additionally, we assessed whether FMT treatment in the absence of oxaliplatin exposure led to similar changes (Figure S3(a)). Although there was no difference in body weight between the control-FMT (FMT only, no oxaliplatin treatment) group and OXA-FMT (FMT only, no oxaliplatin treatment) group (Figure S3(b)), flow cytometry analysis of splenocytes showed that the changes of F4/80⁺ IL-10⁺ macrophages, CD4⁺ IL-10⁺ T cells, and CD4⁺Foxp3⁺ IL-10⁺ regulatory T cells in splenocyte were consistent with the FMT-OXA-exposure experiment results (Figure 5(b)).
我们接着通过流式细胞术确定 IL-10 分泌的来源。脾细胞的流式细胞术分析显示，与对照-FMT 组相比，OXA-FMT 组受体小鼠中 F4/80⁺ IL-10⁺巨噬细胞显著减少（）。有趣的是，脾细胞中 CD4⁺ IL-10⁺ T 细胞和 CD4⁺ Foxp3⁺ IL-10⁺调节性 T 细胞在这两组之间相似（）。此外，我们评估了在未接受奥沙利铂暴露的情况下，FMT 处理是否导致类似变化（图 S3(a)）。尽管对照-FMT 组（仅 FMT，无奥沙利铂治疗）与 OXA-FMT 组（仅 FMT，无奥沙利铂治疗）之间体重无差异（图 S3(b)），脾细胞的流式细胞术分析显示 F4/80⁺ IL-10⁺巨噬细胞、CD4⁺ IL-10⁺ T 细胞及 CD4⁺ Foxp3⁺ IL-10⁺调节性 T 细胞的变化与 FMT-OXA 暴露实验结果一致（）。

To assess the impact of microbiota on macrophage-derived IL-10 secretion, we isolated splenocytes from Rag1^-/- mice and co-cultured them for 24 h with fecal supernatants obtained from control or oxaliplatin-treated mice. Flow cytometry analysis revealed that the number of F4/80⁺ IL-10⁺ cells significantly decreased after treatment with the fecal supernatant from oxaliplatin-treated mice (Figure 5(c)). To further confirm the role of macrophage-derived IL-10 in chemotherapy-induced toxicity, we further established mice model with adoptive transfer of macrophage. F4/80⁺ macrophages were isolated from Il10^-/- and WT mice respectively. These isolated macrophages were transferred into Il10^-/- mice followed by twice of oxaliplatin treatment (Figure 5(d)). Compared with the Il10^-/- mice with F4/80⁺IL-10^−/− macrophages, mice adopted with F4/80⁺ IL-10^+/+ macrophages exhibited a significantly improved weight loss and clinical scores after high dose of oxaliplatin treatment (Figure 5(e,f)). Similar to the phenotype changes, histological analysis also revealed the improved toxicity in the gastrointestinal systems in Il10^-/- mice with F4/80⁺ IL-10^+/+ macrophages adoption (Figures 5(g)).
为了评估微生物群对巨噬细胞来源 IL-10 分泌的影响，我们从 Rag1 ^-/- 小鼠中分离脾细胞，并与来自对照组或奥沙利铂处理小鼠的粪便上清液共同培养 24 小时。流式细胞术分析显示，经过奥沙利铂处理小鼠粪便上清液处理后，F4/80 ⁺ IL-10 ⁺ 细胞数量显著减少（ Figure 5(c) ）。为了进一步确认巨噬细胞来源 IL-10 在化疗诱导毒性中的作用，我们建立了巨噬细胞过继转移的小鼠模型。分别从 Il10 ^-/- 和野生型（WT）小鼠中分离 F4/80 ⁺ 巨噬细胞。将这些分离的巨噬细胞转移至 Il10 ^-/- 小鼠体内，随后进行两次奥沙利铂治疗（ Figure 5(d) ）。与接受 F4/80 ⁺ IL-10 ^−/− 巨噬细胞的 Il10 ^-/- 小鼠相比，接受 F4/80 ⁺ IL-10 ^+/+ 巨噬细胞过继的小鼠在高剂量奥沙利铂治疗后表现出显著改善的体重减轻和临床评分（ Figure 5(e,f) ）。与表型变化相似，组织学分析也显示，接受 F4/80 ⁺ IL-10 ^+/+ 巨噬细胞过继的 Il10 ^-/- 小鼠胃肠系统毒性明显改善（ Figures 5(g) ）。

To explore the downstream changes in macrophage, we next analyzed the splenic transcriptome. Differential genes associated with microbial antigen presentation, such as Toll-like receptor 4 (TLR4), TLR9, TLR12, CD40, CCL4, and CARD11, were significantly downregulated in the OXA-FMT group compared to control-FMT group (Figure S3(c)). Downstream signaling molecules associated with the NF-κB signaling pathway, such as IKBKB, IKBKG, TNFSF14, TRAF3, and TRAF5, were consistently downregulated (Figure S3(c)). These alteration was validated by qPCR analysis (Figure S3(d)). Specifically, TLR4 signaling plays an essential role in bacteria-induced innate immune responses. Similar to the splenic transcriptome, mRNA expression of TLR4, Myd88, NFKB1A and IL-10 was significantly downregulated in RAW264.7 murine macrophage cell line and bone marrow-derived macrophages (BMDMs) stimulated with fecal supernatant from oxaliplatin-treated mice (Figures S3(e-f)). To further confirm the role of TLR4 in IL-10 secretion, we isolated splenocytes from tlr4^Lps-del mice in which harbored dysfunction of TLR4. These isolated splenocytes were exposed to fecal supernatant from oxaliplatin-treated mice. The number of F4/80⁺ IL-10⁺ cells was significantly decreased in tlr4^Lps-del mice compared to that in WT mice (Figure S3(g,h)). These data suggest that microbiota-mediated chemotherapy-induced toxicity is associated with the suppression of TLR4-IL-10 signaling pathway in macrophage.
为了探究巨噬细胞的下游变化，我们接着分析了脾脏转录组。与微生物抗原呈递相关的差异基因，如 Toll 样受体 4（TLR4）、TLR9、TLR12、CD40、CCL4 和 CARD11，在 OXA-FMT 组中相比对照-FMT 组显著下调（图 S3(c)）。与 NF-κB 信号通路相关的下游信号分子，如 IKBKB、IKBKG、TNFSF14、TRAF3 和 TRAF5，也持续下调（图 S3(c)）。这些变化通过 qPCR 分析得到了验证（图 S3(d)）。具体而言，TLR4 信号在细菌诱导的先天免疫反应中起着关键作用。与脾脏转录组类似，使用来自奥沙利铂处理小鼠的粪便上清液刺激的 RAW264.7 小鼠巨噬细胞系和骨髓来源巨噬细胞（BMDMs）中，TLR4、Myd88、NFKB1A 和 IL-10 的 mRNA 表达显著下调（图 S3(e-f)）。为进一步确认 TLR4 在 IL-10 分泌中的作用，我们分离了携带 TLR4 功能障碍的 tlr4^0 小鼠的脾细胞，并用来自奥沙利铂处理小鼠的粪便上清液处理这些分离的脾细胞。 与野生型小鼠相比，tlr4 ^Lps-del 小鼠中 F4/80 ⁺ IL-10 ⁺ 细胞数量显著减少（图 S3(g,h)）。这些数据表明，微生物群介导的化疗诱导毒性与巨噬细胞中 TLR4-IL-10 信号通路的抑制有关。

Chemotherapy-induced toxicity is associated with depletion of bacteria with probiotic properties
化疗诱导的毒性与具有益生菌特性的细菌耗竭相关。

Our results suggested that oxaliplatin causes cellular toxicity by impairing microbiota-induced IL-10 expression in macrophages. To gain more insight into microbiota alterations, we determined the bacterial composition in mice treated with oxaliplatin or PBS using 16S rRNA sequencing. Compared with the microbial feature in baseline and control group, our taxonomic analysis of the microbiome using principal coordinate analysis (PCoA) showed a significant clustering and separation in mice treated with oxaliplatin (Figure 6(a)). We subsequently detected marked differences in the bacterial community abundance after oxaliplatin treatment. Notably, several genera with probiotic properties, such as Lactobacillus (Limosilactobacillus and Ligilactobacillus), Bifidobacterium and Blautia were significantly depleted in oxaliplatin-treated mice, while some genera including Ruminococcus, Paramuribaculum and Clostridium were enriched in oxaliplatin-treated mice (Figure 6(b)). We subsequently applied qPCR analysis to validate the changes of probiotics. Our results confirmed that the relative abundance of Lactobacillus and Bifidobacterium were lower in the feces of oxaliplatin-treated mice and recipients that received FMT from oxaliplatin-treated donors (Figures 6(c,d)). These data demonstrate that the toxicity of chemotherapy is associated with the depletion of bacteria with potential probiotic functions.
我们的结果表明，奥沙利铂通过抑制巨噬细胞中微生物群诱导的 IL-10 表达，导致细胞毒性。为了深入了解微生物群的变化，我们使用 16S rRNA 测序分析了接受奥沙利铂或 PBS 处理的小鼠的细菌组成。与基线和对照组的微生物特征相比，我们通过主坐标分析（PCoA）对微生物组的分类学分析显示，奥沙利铂处理的小鼠表现出显著的聚类和分离（ Figure 6(a) ）。随后，我们检测到奥沙利铂处理后细菌群落丰度的显著差异。值得注意的是，具有益生菌特性的几个属，如乳酸杆菌属（Limosilactobacillus 和 Ligilactobacillus）、双歧杆菌属和布劳氏菌属，在奥沙利铂处理的小鼠中显著减少，而一些属如瘤胃球菌属、Paramuribaculum 属和梭菌属在奥沙利铂处理的小鼠中则显著富集（ Figure 6(b) ）。随后，我们应用 qPCR 分析验证了益生菌的变化。 我们的结果证实，奥沙利铂处理小鼠及接受来自奥沙利铂处理供体的粪菌移植（FMT）受体的粪便中，乳酸杆菌和双歧杆菌的相对丰度较低（ Figures 6(c,d ）。这些数据表明，化疗的毒性与具有潜在益生菌功能的细菌的减少有关。

Restoration of microbiota-depleted probiotics alleviates chemotherapy-induced toxicity
恢复微生物群缺失的益生菌可缓解化疗引起的毒性反应

To demonstrate the importance of microbiota-depleted probiotics in oxaliplatin-induced toxicity, we isolated three strains of probiotics (Bifidobacterium longum, Lactobacillus reuteri, and Lactobacillus johnsonii) from healthy volunteers and gavaged mice with this mixture after a 5-days treatment regimen of an antibiotic cocktail (Figure 6(e)). Importantly, treatment with these probiotics significantly alleviated weight loss and reduced the clinical score of toxicity in mice following chemotherapy exposure (Figures 6(f–g)). Overall survival was also significantly improved in mice treated with the probiotics mixture (Figure 6(h)). Histological analysis further revealed improved toxicity in the hematopoietic and gastrointestinal systems (Figures 6(i–k)). Moreover, the mRNA expression of pro-inflammatory molecules (IL-1β, IL-6, and TNF-α) was significantly decreased, whereas the expression of epithelial barrier molecules (claudin and occludin) was significantly increased in the colon (Figure S4(a)). TLR4 signaling-associated molecules were also significantly upregulated in splenocytes from probiotics-treated mice (Figure S4(b)). Notably, probiotics treatment significantly increased the percentage of F4/80⁺ IL-10⁺ macrophages in the spleen (Figure S4(c)). In addition, IHC results showed a significant increase in the proportion of macrophages and IL-10 expression in the colon, spleen, and bone marrow tissues from mice treated with probiotics (Figures 6(l-n)). Similar alleviation of chemotherapy-induced toxicity was also found in mice treated with a mixture of these three probiotics without antibiotic cocktail pre-treatment (Figures S4(d-f)). More importantly, significant improvement of chemotherapy-induced toxicity was further observed in tumor-bearing mice model after treatment of these three probiotics (Figures S4(g-n)). These findings indicate that supplementation with probiotics can improve chemotherapy-induced toxicity.
为了证明缺失微生物群益生菌在奥沙利铂诱导毒性中的重要性，我们从健康志愿者中分离出三株益生菌（长双歧杆菌、瑞特氏乳杆菌和约翰逊乳杆菌），并在经过 5 天抗生素混合物治疗方案后对小鼠进行灌胃（ Figure 6(e) ）。重要的是，使用这些益生菌治疗显著缓解了小鼠化疗暴露后的体重减轻，并降低了毒性的临床评分（ Figures 6(f–g ）。接受益生菌混合物治疗的小鼠总体存活率也显著提高（ Figure 6(h) ）。组织学分析进一步显示造血系统和胃肠系统的毒性得到改善（ Figures 6(i–k ）。此外，结肠中促炎分子（IL-1β、IL-6 和 TNF-α）的 mRNA 表达显著降低，而上皮屏障分子（claudin 和 occludin）的表达显著增加（图 S4(a)）。益生菌处理小鼠的脾细胞中与 TLR4 信号通路相关的分子也显著上调（图 S4(b)）。 值得注意的是，益生菌治疗显著增加了脾脏中 F4/80⁺ IL-10⁺巨噬细胞的比例（图 S4(c)）。此外，免疫组化（IHC）结果显示，益生菌处理小鼠的结肠、脾脏和骨髓组织中巨噬细胞比例及 IL-10 表达显著增加（图 S4(d-f)）。在未进行抗生素混合物预处理的情况下，使用这三种益生菌混合物治疗的小鼠也表现出类似的化疗诱导毒性缓解（图 S4(d-f)）。更重要的是，在肿瘤负荷小鼠模型中，经过这三种益生菌治疗后，化疗诱导的毒性显著改善（图 S4(g-n)）。这些发现表明，补充益生菌能够改善化疗诱导的毒性。

Enrichment of short-chain fatty acids by probiotics improves chemotherapy-induced toxicity
益生菌富集短链脂肪酸改善化疗诱导的毒性

Previous studies have demonstrated that probiotics favor the production of short-chain fatty acids (SCFAs).¹⁷ Butyrate engages dendritic cells and macrophages to promote IL-10 secretion.¹⁸ Next, we performed targeted metabolome analysis of the feces of mice administered probiotics. As expected, seven SCFAs, including butyrate, were significantly enriched in the mice treated with the probiotics mixture (Figure S5(a)). Additionally, flow cytometry analysis showed that probiotics supernatant increased the proportion of F4/80⁺ IL-10⁺ macrophages in Rag1^-/- mice (Figure S5(b)). To confirm the beneficial role of SCFAs in alleviating chemotherapy-induced toxicity, mice challenged with oxaliplatin were gavaged with butyrate or PBS (Figure S5(c)). Consistent with the above results, the weight loss and the clinical score of toxicity in mice treated with butyrate were significantly improved, as well as the toxicity of the hematopoietic and gastrointestinal systems (Figures S5(d–h)). Furthermore, IHC results showed a significant increase in the proportion of macrophages and IL-10 expression in the colon, spleen, and bone marrow tissues of mice treated with butyrate (Figures S5(i–k). These data suggest that the alleviation of chemotherapy-induced toxicity by probiotics is associated with the production of SCFAs.
先前的研究表明，益生菌有利于短链脂肪酸（SCFAs）的产生。 ¹⁷ 丁酸能够激活树突状细胞和巨噬细胞，促进 IL-10 的分泌。 ¹⁸ 接下来，我们对给予益生菌的小鼠粪便进行了靶向代谢组分析。正如预期，七种短链脂肪酸，包括丁酸，在接受益生菌混合物处理的小鼠中显著富集（图 S5(a)）。此外，流式细胞术分析显示，益生菌上清液增加了 Rag1 ^-/- 小鼠中 F4/80 ⁺ IL-10 ⁺ 巨噬细胞的比例（图 S5(b)）。为了确认短链脂肪酸在缓解化疗诱导毒性中的有益作用，给予奥沙利铂的小鼠通过灌胃给予丁酸或 PBS（图 S5(c)）。与上述结果一致，接受丁酸处理的小鼠体重减轻和毒性临床评分显著改善，造血系统和胃肠系统的毒性也得到缓解（图 S5(d–h)）。此外，免疫组化（IHC）结果显示，接受丁酸处理的小鼠结肠、脾脏和骨髓组织中巨噬细胞比例及 IL-10 表达显著增加（图 S5(i–k)）。 这些数据表明，益生菌缓解化疗引起的毒性与短链脂肪酸（SCFAs）的产生有关。

Improvement of toxicity does not influence efficacy of chemotherapy
毒性的改善不影响化疗的疗效

To evaluate the efficacy of chemotherapy after amelioration of toxicity, we orally administered probiotics and intraperitoneally injected oxaliplatin into mice that subcutaneously harbored MC38 CRC cells (Figure 7(a)). Mice treated with chemotherapy exhibited slower tumor growth, as well as a corresponding reduction in tumor size and weight (Figures 7(b–d)). Notably, probiotics administration had no significant effect on the therapeutic efficacy of the chemotherapy. As an important cytokine in chemotherapy toxicity, rIL-10 injection in combination with oxaliplatin was subsequently administered to mice harboring subcutaneous MC38 cells (Figure 7(e)). Administration of IL-10 did not influence the efficacy of chemotherapy (Figures 7 (f-h)). These data indicate that the amelioration of chemotherapy-induced toxicity by probiotics or IL-10 does not influence the efficacy of chemotherapy.
为了评估缓解毒性后化疗的疗效，我们口服给予益生菌，并腹腔注射奥沙利铂于皮下移植有 MC38 结直肠癌细胞的小鼠（ Figure 7(a) ）。接受化疗的小鼠表现出肿瘤生长减缓，以及肿瘤体积和重量的相应减少（ Figures 7(b–d) ）。值得注意的是，益生菌的给药对化疗的治疗效果无显著影响。作为化疗毒性中的重要细胞因子，随后将重组 IL-10（rIL-10）与奥沙利铂联合注射于皮下移植有 MC38 细胞的小鼠（ Figure 7(e) ）。IL-10 的给药未影响化疗的疗效（ Figures 7 (f-h) ）。这些数据表明，益生菌或 IL-10 缓解化疗引起的毒性不影响化疗的疗效。

To further verify the association between IL-10 and chemotherapy, we established a clinical cohort of CRC patients exposed to oxaliplatin neoadjuvant chemotherapy. The number of CD45⁺IL-10⁺ cells in the peripheral blood mononuclear cells (PBMCs) of CRC patients who did not receive chemotherapy was significantly higher than that in patients who received chemotherapy (Figures 7(i,j)). Importantly, CD45⁺IL-10⁺ cells in the peripheral blood of patients with CRC were significantly suppressed after treatment with chemotherapy (Figure 7(k)). Moreover, re-analysis of the public dataset PRJEB35526⁸ in children with acute lymphoblastic leukemia (ALL) showed a significant decrease in Bifidobacterium after two or three weeks of chemotherapy, which was associated with the occurrence of gastrointestinal toxicity (Figure 7(l)). These data observed in clinical cohorts confirmed an impaired IL-10 levels and a decrease in probiotics strains upon chemotherapy, suggesting potential new therapeutic targets for chemotherapy-induced toxicity.
为了进一步验证 IL-10 与化疗之间的关联，我们建立了一个接受奥沙利铂新辅助化疗的结直肠癌（CRC）患者临床队列。未接受化疗的 CRC 患者外周血单个核细胞（PBMCs）中 CD45^+ IL-10^+细胞的数量显著高于接受化疗的患者（图 2）。重要的是，CRC 患者外周血中的 CD45^+ IL-10^+细胞在化疗治疗后显著减少（图 5）。此外，对儿童急性淋巴细胞白血病（ALL）公共数据集 PRJEB35526 的重新分析显示，经过两到三周的化疗后，双歧杆菌显著减少，这与胃肠道毒性的发生相关（图 7）。这些临床队列中观察到的数据证实了化疗导致 IL-10 水平受损和益生菌菌株减少，提示了化疗诱导毒性的潜在新治疗靶点。

Mice  小鼠

Six-to eight-week-old male C57BL/6 and IL-10^-/-, Rag1^-/-, and tlr4^Lps-del mice were purchased from GemPharmatech. All mice were housed under a 12 h light-dark cycle in an SPF facility and fed a sterilized laboratory rodent diet, 5L0D (LabDiet).
6 至 8 周龄的雄性 C57BL/6、IL-10 ^-/- 、Rag1 ^-/- 和 tlr4 ^Lps-del 小鼠购自 GemPharmatech。所有小鼠均在 SPF 设施中，12 小时光暗循环下饲养，喂食灭菌的实验室啮齿动物饲料 5L0D（LabDiet）。

Bacterial strains  细菌菌株

Lactobacillus reuteri, Lactobacillus johnsonii, and Bifidobacterium longum were isolated from healthy individuals and identified via 16S rRNA sequencing. All strains were grown at 37°C under anaerobic conditions in de Man, Rogosa, and Sharpe (MRS) medium.
从健康个体中分离出乳杆菌属的瑞特氏乳杆菌、约翰逊乳杆菌和双歧杆菌属的长双歧杆菌，并通过 16S rRNA 测序进行鉴定。所有菌株均在 37°C 厌氧条件下于 de Man、Rogosa 和 Sharpe（MRS）培养基中培养。

Human samples  人体样本

Peripheral blood samples were collected before chemotherapy or after the fourth cycle of chemotherapy in CRC patients.
在结直肠癌患者化疗前或第四个化疗周期后采集外周血样本。

Oxaliplatin intervention
奥沙利铂干预

A toxic dose of oxaliplatin (20 mg/kg body weight) was administered to mice via peritoneal injection. The mice were then housed in sterile autoclaved cages and provided standard chow and water ad libitum, unless otherwise noted. The mice were monitored for changes in body weight and other body parameters after the injection, unless otherwise noted. Clinical scores were determined using a cumulative scoring system (Supplementary Table S1), based on weight loss, temperature changes, physical appearance, posture, and mobility.¹⁵ Half of the serum from survived mice were used for the detection of routine blood parameters and another half of the serum from survival mice were used for the detection of oxaliplatin concentration. In the tumor-bearing mouse model, standard treatment doses (10 mg/kg body weight) or toxic doses (20 mg/kg body weight) of oxaliplatin were administered via peritoneal injection. The size and shape of the tumors were monitored every two days.
通过腹腔注射给予小鼠毒性剂量的奥沙利铂（20 mg/kg 体重）。随后将小鼠安置在无菌高压灭菌笼中，自由摄取标准饲料和饮水，除非另有说明。注射后监测小鼠体重及其他身体参数的变化，除非另有说明。临床评分采用累积评分系统（补充表 S1），基于体重减轻、体温变化、外观、姿势和活动能力进行评定。 ¹⁵ 存活小鼠的血清一半用于常规血液参数检测，另一半用于奥沙利铂浓度检测。在肿瘤负荷小鼠模型中，通过腹腔注射给予标准治疗剂量（10 mg/kg 体重）或毒性剂量（20 mg/kg 体重）的奥沙利铂。肿瘤的大小和形状每两天监测一次。

Probiotics treatment experiment
益生菌治疗实验

All SPF C57BL/6 or Il10^-/- mice (male, 6–8 weeks old) were treated with a broad-spectrum antibiotic cocktail (ampicillin 0.2 g/L, metronidazole 0.2 g/L, neomycin 0.2 g/L, and vancomycin 0.1 g/L) in drinking water for five days. For probiotics colonization experiments, after a one-day washout period, mice were orally gavaged with a mixture of probiotics (1 × 10⁹ CFU/dose) or PBS thrice weekly, followed by oxaliplatin intervention.
所有 SPF C57BL/6 或 Il10 ^-/- 小鼠（雄性，6–8 周龄）饮用水中添加广谱抗生素混合液（氨苄青霉素 0.2 g/L、甲硝唑 0.2 g/L、新霉素 0.2 g/L 和万古霉素 0.1 g/L）处理五天。益生菌定植实验中，经过一天的清除期后，小鼠口服灌胃益生菌混合物（1 × 10 ⁹ CFU/剂量）或 PBS，每周三次，随后进行奥沙利铂干预。

Liquid chromatograph mass spectrometer (LC-MS) analysis
液相色谱质谱联用仪（LC-MS）分析

For the serum samples, 50 µL samples were mixed with 300 µL mass spectrometry grade pre-chilled acetonitrile, then vortexed for 5 min. The mixture was then centrifuged at 15,000 × g and 4°C for 10 minutes, and the supernatant was collected. For fecal samples, 20 mg samples were weighed into a 2-mL screw top tube containing 50 mg of acid-washed glass beads, and then 120 µL mass spectrometry grade pre-chilled acetonitrile was added to each tube. The samples were homogenized under 70 Hz cryogenic grinding for 5 min. The tubes were then centrifuged at 15,000 × g and 4°C for 10 min, and the supernatant was collected. Measurements were obtained using an Agilent 1290 Infinity II Liquid Chromatography System coupled to an Agilent 6495A Triple Quadrupole Liquid Chromatography-Mass Spectrometry (LC-MS) System. Data analysis was conducted using MassHunter Workstation Data Acquisition, Agilent MassHunter VistaFlux Software, and Agilent Metabolite ID Software. The metabolites were identified based on the standards, MS/MS spectra, and the metabolite database METLIN (https://metlin.scripps.edu/indexphp).
对于血清样本，取 50 µL 样本与 300 µL 质谱级预冷乙腈混合，然后涡旋振荡 5 分钟。混合物随后在 15,000 × g、4°C 下离心 10 分钟，收集上清液。对于粪便样本，称取 20 mg 样本置于含有 50 mg 酸洗玻璃珠的 2 mL 螺旋盖管中，然后向每管中加入 120 µL 质谱级预冷乙腈。样本在 70 Hz 冷冻研磨条件下均质 5 分钟。随后管子在 15,000 × g、4°C 下离心 10 分钟，收集上清液。测量使用安捷伦 1290 Infinity II 液相色谱系统联用安捷伦 6495A 三重四极杆液相色谱-质谱（LC-MS）系统进行。数据分析采用 MassHunter 工作站数据采集软件、安捷伦 MassHunter VistaFlux 软件及安捷伦代谢物识别软件。代谢物鉴定基于标准品、MS/MS 光谱及代谢物数据库 METLIN（ https://metlin.scripps.edu/indexphp ）。

Fecal microbiota transplantation
粪便微生物移植

SPF C57BL/6 donor mice were injected with oxaliplatin (20 mg/kg body weight) or PBS for two weeks. Fecal pellets (200–250 mg) were collected in sterile tubes prior to suspension and homogenization in 2 mL of PBS. After centrifugation at 100 × g at 4°C for 30 s, bacteria-enriched supernatants were collected and transplanted into mice (200 μL per mouse) by oral gavage three times weekly. Recipient mice were treated with an antibiotic cocktail for five days and a one-day washout period, followed by FMT intervention (three times a week).
SPF C57BL/6 供体小鼠连续两周注射奥沙利铂（20 mg/kg 体重）或 PBS。收集粪便颗粒（200–250 mg）于无菌管中，悬浮并匀浆于 2 mL PBS 中。4°C 下以 100 × g 离心 30 秒后，收集富含细菌的上清液，通过口服灌胃将其移植到小鼠体内（每只小鼠 200 μL），每周三次。受体小鼠先用抗生素混合物处理五天，停药一天后进行粪菌移植干预（每周三次）。

Macrophage depletion experiment
巨噬细胞清除实验

Mice were treated with an antibiotic cocktail for five days, after a one-day washout period, followed by intraperitoneal injection of clodronate liposomes or control liposomes (FormuMax) (200 μL per mouse) to eliminate macrophages. Subsequently, the FMT experiment was conducted for two weeks, as previously described. Mice were exposed to a high dose of oxaliplatin (20 mg/kg body weight).
小鼠先用抗生素混合物处理五天，停药一天后，腹腔注射氯膦酸脂质体或对照脂质体（FormuMax）（每只小鼠 200 μL）以清除巨噬细胞。随后，按照前述方法进行为期两周的粪菌移植实验。小鼠接受高剂量奥沙利铂（20 mg/kg 体重）处理。

rIL-10 and SCFA treatment
重组 IL-10 和短链脂肪酸（SCFA）治疗

Mice were intraperitoneally injected with rIL-10 (100 ng/mouse/injection in 0.1 mL of PBS; Novoprotein) twice a week during the course of oxaliplatin intervention. Sodium butyrate (200 mM) was administered to the mice in drinking water for two weeks, followed by oxaliplatin treatment.
在奥沙利铂干预期间，小鼠腹腔注射重组 IL-10（rIL-10，100 ng/只/次，溶于 0.1 mL PBS；Novoprotein），每周两次。丁酸钠（200 mM）通过饮水给予小鼠两周，随后进行奥沙利铂治疗。

Macrophage isolation and adoptive transfer
巨噬细胞分离与过继转移

Donor mice were sacrificed and the spleen was harvested. Spleen immune cell was isolated and macrophages were further isolated by using magnetic bead separation methods. In short, the cell number in the single cell suspension was determined and then centrifuged. Next, the cell pellet was incubated with anti-F4/80 microbeads (130-110-443, Miltenyi Biotec) according to the manufacturer’s instructions. Recipient mice were injected intravenously with 2 × 10⁶ macrophages. Then mice were treated with oxaliplatin after three days of injection.
供体小鼠被处死，脾脏被取出。分离脾脏免疫细胞后，采用磁珠分离法进一步分离巨噬细胞。简而言之，首先测定单细胞悬液中的细胞数目并离心。然后，将细胞沉淀按照制造商说明与抗 F4/80 磁珠（130-110-443，Miltenyi Biotec）孵育。受体小鼠静脉注射 2 × 10^7 个巨噬细胞。注射三天后，小鼠接受奥沙利铂治疗。

Tumor inoculation  肿瘤接种

Mice were subcutaneously inoculated with 10⁶ MC38 cells in the abdominal flank. Tumor volume was measured every two days and calculated using the formula (length × width² ×0.5. Five days after tumor inoculation, oxaliplatin (10 mg/kg body weight) was administered to mice twice a week. For probiotic treatment, mice were gavaged with a mixture of probiotics (1 × 10⁹ CFU/dose) or PBS thrice weekly before tumor inoculation. For rIL-10 treatment, the mice were injected with rIL-10 twice before tumor inoculation.
小鼠在腹侧皮下接种 10^6 个 MC38 细胞。肿瘤体积每两天测量一次，计算公式为（长度 × 宽度 × 0.5）。肿瘤接种五天后，给予小鼠每公斤体重 10 mg 的奥沙利铂，每周两次。益生菌治疗中，小鼠在肿瘤接种前每周三次灌胃益生菌混合物（1 × 10^8 CFU/剂量）或 PBS。重组 IL-10（rIL-10）治疗中，小鼠在肿瘤接种前注射两次 rIL-10。

High throughput 16S rRNA amplicon sequencing and analysis
高通量 16S rRNA 扩增子测序及分析

Genomic DNA was extracted using a FastDNA Spin Kit for Soil (MP Biomedicals). For 16S rRNA gene sequencing, the V3-V4 variable region was amplified using 2-step PCR. In the first step, 10 ng genomic DNA was used as a template for the first PCR with a total volume of 20 μl using the 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806 R (5’-GGACTACHVGGGTWTCTAAT-3’) primers appended with Illumina adaptor sequences. The amplicons were purified, checked on a fragment analyzer, quantified, followed by equimolar multiplexing, and sequenced on an Illumina MiSeq PE300 platform. The pooled amplicons were further qualified and quantified using the Microbial Ecology 2 (QIIME2) software. Reads were imported, quality-filtered, and dereplicated with the q2-data2 plugin. Subsequently, the dada2 plugin was used with paired-end reads, with truncation of the primer sequences and trimming of the reads. The sequences were classified using Greengenes2⁴² as the reference 16S rRNA gene database. PCoA, LEfSe, and significant species were analyzed using R (v4.1.1).⁴³
使用 FastDNA 土壤旋转试剂盒（MP Biomedicals）提取基因组 DNA。对于 16S rRNA 基因测序，采用两步 PCR 扩增 V3-V4 可变区。第一步中，使用 10 ng 基因组 DNA 作为模板，采用 338F（5’-ACTCCTACGGGAGGCAGCAG-3’）和 806R（5’-GGACTACHVGGGTWTCTAAT-3’）引物（附带 Illumina 接头序列），反应体系总量为 20 μl 进行首次 PCR 扩增。扩增产物经纯化、片段分析仪检测、定量后，按等摩尔混合，随后在 Illumina MiSeq PE300 平台上进行测序。混合的扩增产物进一步使用 Microbial Ecology 2（QIIME2）软件进行质量鉴定和定量。利用 q2-data2 插件导入序列，进行质量过滤和去重复。随后，使用 dada2 插件处理双端序列，截断引物序列并修剪序列。序列分类采用 Greengenes2 作为参考 16S rRNA 基因数据库。主坐标分析（PCoA）、线性判别分析效应大小（LEfSe）及显著物种分析均使用 R 软件（v4.1.1）完成。 ⁴³

Host RNA sequencing and analysis
宿主 RNA 测序及分析

Splenic samples were obtained from mice subjected to FMT. Total RNA was extracted from splenic tissues using TRIzol Reagent (Invitrogen), according to the manufacturer’s instructions (Invitrogen). RNA integrity was evaluated using ND-2000 (NanoDrop Technologies, USA) and 2100 Bioanalyzer (Agilent Technologies). RNA-seq libraries were prepared using the TruSeq RNA Sample Prep kit (Illumina), and libraries were successfully constructed from splenic samples. Briefly, messenger RNA was isolated according to the polyA selection method using oligo (dT) beads, and then fragmented using fragmentation buffer. Double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. Libraries were selected for cDNA target fragments of 300 bp on 2% low range ultra-agarose, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification using TBS380, the paired-end RNA-seq sequencing library was sequenced using the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150bp read length). Differential expression analysis between the two groups was performed using the Limma R package. Genes with an adjusted p-value <0.05 and |Log2(Fold Change)|＞0 were assigned as significantly differentially expressed.
从接受粪菌移植（FMT）的小鼠中获取脾脏样本。按照制造商说明（Invitrogen）使用 TRIzol 试剂（Invitrogen）提取脾脏组织中的总 RNA。使用 ND-2000（NanoDrop Technologies，美国）和 2100 生物分析仪（Agilent Technologies）评估 RNA 完整性。使用 TruSeq RNA 样本制备试剂盒（Illumina）构建 RNA 测序文库，脾脏样本成功构建文库。简要地，采用寡聚(dT)磁珠根据 polyA 选择法分离信使 RNA，然后使用片段化缓冲液进行片段化。使用 SuperScript 双链 cDNA 合成试剂盒（Invitrogen）和随机六聚体引物（Illumina）合成双链 cDNA。随后，按照 Illumina 文库构建协议对合成的 cDNA 进行末端修复、磷酸化和加“A”碱基。文库在 2%低范围超琼脂糖凝胶上选择 300 bp 的 cDNA 目标片段，随后使用 Phusion DNA 聚合酶（NEB）进行 15 个 PCR 循环扩增。 使用 TBS380 进行定量后，采用 Illumina HiSeq xten/NovaSeq 6000 测序仪（2 × 150bp 读长）对双端 RNA-seq 测序文库进行测序。两组间的差异表达分析使用 Limma R 包进行。调整后的 p 值<0.05 且|Log2(倍数变化)|＞0 的基因被认定为显著差异表达基因。

Measurement of serum cytokine levels using multiplex immunoassays
使用多重免疫测定法测量血清细胞因子水平

A total of 31 serum cytokines were detected simultaneously using the Bio-Plex Pro Human Cytokine Screening Panel (R&D Systems), according to the manufacturer’s protocol. The tests were performed in accordance with the manufacturer’s procedures, and the sample dilution was 1:2, including the standard curve and blank value. The assay plate was analyzed using a Luminex X-200instrument (Bio-Rad Laboratories). Data were calculated using the Bio-Plex Manager software ver. 5.0 (Bio-Rad Laboratories).
根据制造商的协议，使用 Bio-Plex Pro 人类细胞因子筛选面板（R&D Systems）同时检测 31 种血清细胞因子。测试按照制造商的操作流程进行，样品稀释比例为 1:2，包括标准曲线和空白值。使用 Luminex X-200 仪器（Bio-Rad Laboratories）分析检测板。数据使用 Bio-Plex Manager 软件 5.0 版（Bio-Rad Laboratories）计算。

Targeted metabolome of fecal SCFAs
粪便短链脂肪酸（SCFAs）的靶向代谢组分析

SCFAs were extracted from fecal samples (100 mg) in an aqueous solution and analyzed by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A/5975C instrument (BioNovoGene Company). Chromatographic separation was performed on an Agilent HP-5 capillary column. The analytes were quantified using a series of stock solutions under standard conditions. Briefly, 100 mg of fecal samples were weighed and mixed with 1 mL of 0.005 M NaOH solution with 50 μL 2-methyl-butyric acid for 2 min and incubated at 4°C for 2 h. Next, the mixture was centrifuged at 4°C 13,000 rpm for 20 min, and the supernatant was collected. A total of 500 μL of supernatant was added to 300 μL distilled water, 500 μL isopropanol/pyridine solution, and platelet cytotoxic factor solution for derivatization and then extracted with 500 μL n-hexane for further analysis. Agilent HP-5 capillary column (30 cm *0.25 mm *0.25 μm) was used for GC-MS detection. An Agilent MSD ChemStation (E.02.00.493, Agilent Technologies) was used to analyze the data.
短链脂肪酸（SCFAs）从粪便样本（100 mg）中以水溶液形式提取，并使用安捷伦 7890A/5975C 气相色谱-质谱联用仪（BioNovoGene 公司）进行分析。色谱分离采用安捷伦 HP-5 毛细管柱进行。分析物在标准条件下通过一系列标准溶液进行定量。简而言之，称取 100 mg 粪便样本，加入 1 mL 0.005 M NaOH 溶液和 50 μL 2-甲基丁酸，混合 2 分钟后于 4°C 孵育 2 小时。随后，混合液在 4°C 下以 13,000 rpm 离心 20 分钟，收集上清液。取 500 μL 上清液，加入 300 μL 蒸馏水、500 μL 异丙醇/吡啶溶液及血小板细胞毒因子溶液进行衍生化处理，然后用 500 μL 正己烷提取，进行后续分析。GC-MS 检测采用安捷伦 HP-5 毛细管柱（30 cm × 0.25 mm × 0.25 μm）。数据分析使用安捷伦 MSD ChemStation 软件（E.02.00.493，安捷伦技术公司）。

Cell isolation of spleen mononuclear cells
脾脏单核细胞的分离

Spleens were completely isolated from mice and crushed with forceps, and single cells were isolated in PBS using a 70-μm cell strainer. The cells were washed with 1× PBS and centrifuged (100 × g for 5 min), and then red blood cell lysis containing splenocytes was pipetted up. The culture medium was then added to the cells and centrifuged at 100 × g at 4°C for 5 min. Single-cell suspensions were diluted in Roswell Park Memorial Institute (RPMI) medium.
脾脏从小鼠体内完全分离，用镊子捣碎，单细胞通过 70 微米细胞过滤器在 PBS 中分离。细胞用 1× PBS 洗涤并离心（100×g，5 分钟），然后吸取含有脾细胞的红细胞裂解液。随后向细胞中加入培养基，在 4°C 下以 100×g 离心 5 分钟。单细胞悬液用罗斯威尔帕克纪念研究所培养基（RPMI）稀释。

Isolation of BMDMs and PBMCs
骨髓来源巨噬细胞（BMDMs）和外周血单核细胞（PBMCs）的分离

BMDMs were isolated from the femurs and tibias of mice. Cells were differentiated in BMDM media (Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 25 mM l-glutamate, penicillin/streptomycin, and 200 U/mL recombinant mouse M-CSF. On day 7, the cells were challenged with fecal supernatants and allowed to acclimatize for 24 h.
BMDMs 从小鼠的股骨和胫骨中分离。细胞在 BMDM 培养基中分化（Dulbecco 改良鹰培养基（DMEM）、10%胎牛血清（FBS）、25 mM L-谷氨酸、青霉素/链霉素和 200 U/mL 重组小鼠 M-CSF）。第 7 天，细胞用粪便上清液刺激，并允许适应 24 小时。

Approximately 4 mL of human venous blood was collected in heparinized vials and gently inverted. PBMCs were isolated by gradient centrifugation using Ficoll-paque plus (Cytiva). Isolated cells were washed twice with 10 mL sterile FBS-free Roswell Park Memorial Institute (PRMI) medium. The medium was discarded, and the cells were resuspended in sterile PRMI medium.
采集约 4 毫升人静脉血于肝素化采血管中，轻轻颠倒混匀。通过 Ficoll-paque Plus（Cytiva）密度梯度离心分离 PBMCs。分离的细胞用 10 毫升无 FBS 无菌 RPMI 培养基洗涤两次。弃去培养基，细胞重悬于无菌 RPMI 培养基中。

Cell culture and cellular stimulation
细胞培养和细胞刺激

The murine macrophage cell line RAW264.7 was purchased from the American Type Culture Collection and cultured at 37°C in DMEM (Gibco) supplemented with 10% FBS (Gibco) in a 5% CO₂ atmosphere.
小鼠巨噬细胞系 RAW264.7 购自美国典型培养物保藏中心（ATCC），在 37°C、5% CO₂气氛中，使用含 10%胎牛血清（FBS，Gibco）的 DMEM 培养基（Gibco）培养。

For fecal supernatant stimulation experiments, a ratio of 1 mL PBS per 50 mg feces was used for homogeneously making fecal suspensions, centrifuged at 100 × g for 5 min, and the supernatant was collected. The supernatant was passed through a needle filter to remove the microbiota. The cells (RAW264.7, BMDM, and splenocytes) were stimulated by fecal supernatant for 24 h and RNA was extracted for qPCR analysis or cells were collected for flow cytometry.
粪便上清液刺激实验中，采用 1 mL PBS 对 50 mg 粪便的比例制备均匀的粪便悬液，离心 100×g，5 分钟，收集上清液。上清液通过针头过滤器去除微生物。细胞（RAW264.7、骨髓来源巨噬细胞 BMDM 及脾细胞）用粪便上清液刺激 24 小时，提取 RNA 进行 qPCR 分析，或收集细胞进行流式细胞术。

RNA and DNA extraction for qPCR analysis
用于 qPCR 分析的 RNA 和 DNA 提取

RNA was extracted using a Total RNA Kit (R323–01; Vazyme). cDNA was reverse transcribed using Hiscript@ III RT Super Mix with a gDNA wiper (R323–01, Vazyme). Fecal or bacterial DNA was obtained using an AmPure Microbial DNA Kit (D7111, Megan). qPCR was performed on an Applied Biosystems 7500 Real-Time PCR system using SYBR Green real-time PCR master mix (QPK-201; Toyobo). The primer sequences used in this study are listed in Supplementary Table S2.
使用 Total RNA Kit（R323–01；Vazyme）提取 RNA。使用 Hiscript@ III RT Super Mix 含 gDNA 去除剂（R323–01，Vazyme）进行 cDNA 逆转录。粪便或细菌 DNA 使用 AmPure 微生物 DNA Kit（D7111，Megan）提取。qPCR 在 Applied Biosystems 7500 实时 PCR 系统上使用 SYBR Green 实时 PCR 主混合液（QPK-201；Toyobo）进行。本研究使用的引物序列列于补充表 S2 中。

Histopathology  组织病理学

Spleens, femurs, and colon tissues were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. Before paraffin embedding, femurs underwent an additional decalcification step. The slides were stained with hematoxylin and eosin and morphological changes were observed.
收集脾脏、股骨和结肠组织，固定于 10%中性缓冲福尔马林中，包埋于石蜡中并切片。股骨在石蜡包埋前进行了额外的脱钙步骤。切片用苏木精-伊红染色，观察形态学变化。

Flow cytometry analysis  流式细胞术分析

For IL-10 detection, splenocytes were stimulated with PMA, ionomycin, and brefeldin A for 6 h. PBMCs were stimulated with LPS and brefeldin A for 24 h. The cells were resuspended in PBS and Fc receptors were blocked with anti-CD16/32 antibody (#101320, BioLegend). The following antibodies were used: CD4 (#100411, BioLegend), CD11b (#101226, BioLegend), F4/80 (#123122, BioLegend), and CD45 (#304006, BioLegend). For the intracellular marker IL-10 (#505008 and #501404, BioLegend), FOXP3 (#126405, BioLegend) cells were fixed and permeabilized using the FOXP3/Transcription Factor Staining Buffer kit (#00-5523-00, eBioscience) according to the manufacturer’s instructions after surface staining and incubated with the corresponding antibodies. Labeled cells were analyzed using a CytoFLEX flow cytometer (BECKMAN). Gating strategies are shown in Figure S6.
为了检测 IL-10，脾细胞用 PMA、离子霉素和 Brefeldin A 刺激 6 小时。外周血单个核细胞（PBMCs）用 LPS 和 Brefeldin A 刺激 24 小时。细胞重悬于 PBS 中，并用抗 CD16/32 抗体（#101320，BioLegend）阻断 Fc 受体。使用的抗体包括：CD4（#100411，BioLegend）、CD11b（#101226，BioLegend）、F4/80（#123122，BioLegend）和 CD45（#304006，BioLegend）。对于细胞内标志物 IL-10（#505008 和#501404，BioLegend）和 FOXP3（#126405，BioLegend）细胞，在表面染色后，按照厂家说明使用 FOXP3/转录因子染色缓冲液试剂盒（#00-5523-00，eBioscience）进行固定和通透处理，并用相应抗体孵育。标记细胞使用 CytoFLEX 流式细胞仪（BECKMAN）进行分析。门控策略见图 S6。

Immunohistochemical staining
免疫组织化学染色

IL-10, F4/80, Foxp3, and CD4 expression in the spleen, femur, and colon tissues was determined by IHC. In brief, the following steps were performed: paraffin sections dewaxing to water; the antigen was retrieved; the endogenous peroxidase was blocked with hydrogen peroxide solution; 3%BSA was added at room temperature for 30 min; Anti-IL-10 (GB11108–100, Servicebio) antibody (1:500), Anti -F4/80 (GB113373–100, Servicebio) antibody (1:500), Anti -FOXP3 (GB112325–100, Servicebio) antibody (1:500), or Anti-CD4 (GB15064–100, Servicebio) antibody (1:500) was added, and the mix was refrigerated at 4°C overnight; sheep anti-mouse/rabbit IgG was added; we performed staining with diaminobenzidine (DAB) for 5 min and counterstaining with hematoxylin; the slices were then dehydrated and sealed with neutral gum.
通过免疫组化（IHC）检测脾脏、股骨和结肠组织中 IL-10、F4/80、Foxp3 和 CD4 的表达。简要步骤如下：石蜡切片脱蜡至水；抗原修复；用过氧化氢溶液封闭内源性过氧化物酶；室温下加入 3%BSA 封闭 30 分钟；加入抗 IL-10 抗体（GB11108–100，Servicebio）（1:500）、抗 F4/80 抗体（ GB113373 –100，Servicebio）（1:500）、抗 FOXP3 抗体（ GB112325 –100，Servicebio）（1:500）或抗 CD4 抗体（GB15064–100，Servicebio）（1:500），4°C 冷藏过夜；加入羊抗小鼠/兔 IgG；用二氨基联苯胺（DAB）染色 5 分钟，并用苏木素复染；随后切片脱水并用中性胶封片。

The evaluation criteria for IHC experiments were as follows: The IHC results were obtained in a blinded manner (ImageJ Software) using an established pathological scoring system (H-score), and the intensity of staining was scored as 0= negative, 1= weak, 2= moderate, or 3= strong, and for each intensity, the frequency was indicated in percent (in steps of 10). The H-score was then calculated as the sum of 1× frequency of weak staining + 2× frequency of moderate staining + 3× frequency of strong staining.
免疫组化（IHC）实验的评价标准如下：IHC 结果采用盲法获得（使用 ImageJ 软件），并依据既定的病理评分系统（H 评分）进行评估。染色强度评分为 0=阴性，1=弱，2=中等，3=强，每种强度的频率以百分比表示（以 10%的步长）。H 评分计算公式为：弱染色频率×1 + 中等染色频率×2 + 强染色频率×3 的总和。

Quantification and statistical analysis
定量与统计分析

All data are expressed as mean ± SEM unless otherwise stated in the figure legends. Unless otherwise stated in individual method sections above, all statistical analyses were performed using Prism 8 (GraphPad Software). Two-tailed Student’s t-test (parametric) or Mann – Whitney U test (non-parametric) was used. For comparison of more than three groups, statistical analysis was performed using one-way ANOVA (parametric) or Kruskal-Wallis test (non-parametric). All p-values were two-sided, and an adjusted p-value of < 0.05 was considered statistically significant. The details of the statistical tests used and the pooled values for several biological replicates are indicated in the respective figure legends. Statistically significant values are denoted as *p < .05, **p < .01, ***p < .001, and ****p < .0001.
所有数据均以均数 ± 标准误（SEM）表示，除非图例中另有说明。除上述各方法部分另有说明外，所有统计分析均使用 Prism 8（GraphPad 软件）进行。采用双尾 Student’s t 检验（参数检验）或 Mann–Whitney U 检验（非参数检验）。对于三组以上的比较，采用单因素方差分析（ANOVA，参数检验）或 Kruskal-Wallis 检验（非参数检验）。所有 p 值均为双侧，调整后的 p 值 < 0.05 被视为具有统计学意义。所用统计检验的详细信息及多个生物学重复的合并值见各自图例。统计学显著性用 *p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001 表示。