Sexual dimorphism in gut microbiota dictates therapeutic efficacy of intravenous immunoglobulin on radiotherapy complications

Graphical abstract

Highlights

• •Intravenous injection of IVIg alone fights against radiation-induced hematopoietic and GI tract toxicity in female mice only.
• •Abdominal local irradiation induces a sex-specific gut microbiota configuration and small intestinal gene expression signature.
• •IVIg combined with Lachnospiraceae or hypoxanthine, a metabolite produced by Lachnospiraceae, mitigates radiation injuries in male mice.
• •Lachnospiraceae or hypoxanthine replenishment up-regulates radiation-decreased PLD1 expression in small intestine in male mice. In females, radiation exposure increases the intestinal PLD1, silencing PLD1 blunts the radioprotection of IVIg.
• •IVIg performs radioprotection in a sex-dependent manner through Lachnospiraceae/hypoxanthine/PLD1 axis.

Abstract

Introduction

With the mounting number of cancer survivors, the complications following cancer treatment become novel conundrums and starve for countermeasures. Intravenous immunoglobulin (IVIg) is a purified preparation for immune-deficient and autoimmune conditions.

Objectives

Here, we investigated whether IVIg could be employed to fight against radiation injuries and explored the underlying mechanism.

Methods

Hematopoietic or gastrointestinal (GI) tract toxicity was induced by total body or abdominal local irradiation. High-throughput sequencing was performed to analyze the gut microbiota configurations and gene expression profile of small intestine. The untargeted metabolomics of gut microbiome was assessed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. Hydrodynamic-based gene delivery was used to knockdown the target genes in vivo.

Results

Intravenous injection of IVIg protected against radiation-induced hematopoietic and GI tract toxicity in female mice but not in males. IVIg structured sex-characteristic gut microbiota configurations in abdominal irradiated mice. The irradiation enriched gut Lachnospiraceae in female mice but reduced those in males. IVIg injection combined with oral gavage of Lachnospiraceae or its metabolite hypoxanthine, alleviated radiation toxicity in male mice however, Lachnospiraceae or hypoxanthine alone failed to ameliorate the injuries. Abdominal local irradiation drove sex-distinct gene expression signatures in small intestine. Mechanistic investigation showed that replenishment of Lachnospiraceae or hypoxanthine offset abdominal radiation-reduced PLD1 expression in male mice. In females, irradiation elevated PLD1 expression. Deletion of PLD1 in GI tract of female mice erased the radioprotective effects of IVIg.

Conclusion

IVIg battles against radiation injuries in a sex-specific, gut microbiome-dependent way through Lachnospiraceae/hypoxanthine/PLD1 axis. Our findings provide a sex-precise therapeutic avenue to improve the prognosis of cancer patients with radiotherapy in pre-clinical settings.

Introduction

The advancement in delivery technology of radiotherapy, such as stereotactic body radiotherapy and magnetic resonance imaging (MRI)-guided radiotherapy, have transformed radiotherapy into a more precise remedy for cancer patients [1]. However, the technological innovations still fail to address the adverse side effects intertwined with radiotherapy [2]. The complications remain common and intractable in clinical scenario, halting radiotherapy prematurely and degrading patient life quality. Epidemiological survey of systemic therapy and radiotherapy shows that the hematopoietic and gastrointestinal (GI) syndromes rank as the top 2 treatment-related complications with the incidence rate of approximately 20% and 12.6% respectively [3]. Heretofore, with the on-going elevation in 5-year survival rate and number of cancer survivors, starving for safe and effective avenues to protect patients against adverse side effects with cancer therapy becomes a thorny issue in clinical application.

IVIg, a preparation of highly purified immunoglobulins (mostly of the IgG class) pooled from thousands of healthy blood donors per batch, has been proved to be an efficient anti-inflammatory and immunomodulatory agent for multiple diseases [4]. IVIg comprises antigen-binding (Fab) and crystallizable (Fc) fragments, and both two regions implicate in maintaining immune homeostasis. Overall, IVIg modulates the expression and function of Fc receptors, interferes with complement pathway, neutralizes inflammatory cytokines and malgenic antibodies, governs the activation of innate immune cells, effector T cells (TH1 and TH17) and B cells etc [5], [6]. IVIg has been employed as a safe and effective agent to fight against Kawasaki disease, neurodegenerative diseases such as Alzheimer’s disease and Crohn's disease etc [7], [8], [9]. Autoimmune disorder patients with IVIg treatment always experience objective improvements in fibrotic conditions and life quality [10]. The anti-inflammatory role of IVIg is also reflected in lowering inflammation in SARS-COV-2 infection and preventing acute respiratory distress syndrome [11]. Gut microbiota evolves along with host and is an integral part of the individual. The commensal microbiota assists in nutrient decomposition and absorption, immune maturation and response through the mutual interactions with hosts [12], [13]. Dysbiosis in intestinal ecology precipitates the development of heterogeneous diseases, covering inflammatory bowel disease, colorectal cancer and neurodegenerative diseases [14], [15], [16]. The gut microbiota also represents therapeutic and prophylactic implications on multiple diseases [17], [18]. For example, our previous studies identified that fecal microbiota transplantation and gut microorganism-derived metabolites such as valeric acid (VA) or indole 3-propionic acid (IPA) could mitigate radiation toxicity [19], [20], [21].

In the present study, we aimed to explore the effects of IVIg on radiation injuries and found that intravenous injection of IVIg alone protected female mice against hematopoietic and GI tract syndromes, but not in the male counterparts. However, IVIg combined with oral gavage of Lachnospiraceae, an enteric anaerobe, or its metabolite hypoxanthine mitigated the radiation injuries in male mice. Collectively, our findings provide a sex-specific, gut microbiota-dependent option for male and female to fight against radiation toxicity in a pre-clinical setting.

Materials and methods

Mice

Male and female C57BL/6J mice (6–8 week) were purchased from Huafukang Bioscience Co. Inc (Beijing, China). All experimental mice were housed in the specific pathogen free level animal facility. Mice were kept in the same temperature (22 ± 2 ℃) and air humidity controlled (40–70%) animal room with a 12/12-h light/dark cycle and continuous accessed to a standard diet and water.

Experimental group

The experimental mice were divided into groups randomly. (1) Control group: Healthy C57BL/6 mice of both sexes. (2) Total body irradiation (TBI) group: Mice were received total body irradiation. (3) Total abdominal irradiation (TAI) group: Mice were received abdominal local irradiation. (4) IVIg (pre) group: The first time of IVIg treatment was 24 h before irradiation. (5) IVIg (post) group: The first time of IVIg treatment was immediately after irradiation (within 5 min). (6) IVIg + Lachnospiraceae group: The first time of Lachnospiraceae treatment was before irradiation (within 30 min). (7) IVIg + Hypoxanthine (oral gavage) group: The first time of hypoxanthine treatment via oral route was before irradiation (within 30 min). (8) IVIg + Hypoxanthine (injection) group: The first time of hypoxanthine treatment via intraperitoneal injection was before irradiation (within 30 min). (8) IVIg + shRNA group: Mice were injected with shRNA solution and IVIg after TAI. More than 10 mice per group.

Irradiation study

Radiation was delivered using the Gammacell-40 137Cs irradiator (Atomic Energy of Canada Limited, Chalk River, ON, Canada). The dose rate was 0.9 Gy/minute. Mice were received a single dose of 4 Gy γ-ray to perform TBI (for hematopoietic system experiments). Mice were received a single dose of 12 Gy γ-ray to perform TAI for GI tract experiments using a lead shielding so that only whole abdominal was irradiated. Control mice were sham-irradiated.

Intravenous immunoglobulin agent

The Intravenous immunoglobulin agent was obtained from Shanghai RAAS Blood products Co., Ltd., Shanghai, China. IVIg concentration was 50 mg/ml. To maintain plenitudinous blood level of immunoglobulin, the experimental mice were intravenously injected with IVIg 150 µl per 20 g body weight every 3 days and total 5 times based on previous studies [22], [23].

Lachnospiraceae bacterium

Lachnospiraceae bacterium was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM No. 24404). The bacteria were anaerobic cultured in Bugbox plus anaerobic & microaerophilic workstation from Baker Ruskinn at 37 °C with N₂ (90 %), H₂ (10 %) using Columbia agar base with 5% defibrinated sheep blood. The experimental mice were treated with the bacteria via oral route every two days for two weeks.

Hypoxanthine agent

The hypoxanthine (H8010) was purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. Hypoxanthine was suspended using sterilizing water. The experimental mice were treated with hypoxanthine 1 mg per 20 g body weight every 2 days via oral route or intraperitoneal injection for two weeks.

Bacterial diversity analysis

Bacterial configuration analysis was the same as our previous work [21]. Briefly, 16S ribosomal RNA V4 of gut microbiota was amplified. Sequencing libraries were generated by Qubit@ 2.0 Fluorometer (Thermo Scientific), Agilent Bioanalyzer 2100 system were used to evaluate the quality. The library was sequenced on an IlluminaNovaSeq6000 PE250 platform. Sequence analysis was performed by Uparse v7.0.1001 (https://drive5.com/uparse/). Sequences similarity more than 97% were allocated to the same OTUs. The Silva123 Database with RDP classifier (Version 2.2, https://sourceforge.net/projects/rdpclassifier/) algorithm was used to annotate taxonomic information (Novogene Bioinformatics Technology Co., Ltd.). The primers were listed in Supplementary Table 1.

Metabolomics analysis

Gut metabolomics was analyzed as our previously described [24]. Feces from the experimental mice were analyzed by LC-MS/MS using a Vanquish UHPLC system coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol), and eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (Methanol) for the negative polarity mode. The solvent gradient was 2% B, 1.5 min; 2–100 % B, 12.0 min; 100 % B, 14.0 min; 100–2 % B, 14.1 min; 2 % B, 16 min. The raw data were generated by Compound Discoverer 3.0 (CD 3.0, Thermo Fisher). Peaks matched with mzCloud (https://www.mzcloud.org/) and ChemSpider (https://www.chemspider.com/) database were collected as the accurate and relative quantitative results (Novogene Bioinformatics Technology Co., Ltd.).

Transcriptome sequencing

Transcriptome sequencing was analyzed as previously described [25]. Briefly, a sequencing library were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). The library was purified with AMPure XP system (Beckman Coulter, Beverly, USA) to select 250 ∼ 300 bp cDNA fragments. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was evaluated by Agilent Bioanalyzer 2100 system.

Hydrodynamic-based gene delivery

Plasmid constructs: Sh-ASAP1 and sh-PLD1 sequences were forecasted by BLOCK-iT™ RNAi Designer, and the fragments were inserted into the pRNA-U6.1/Neo vector (BamH I and Hind III sites). The plasmids were synthetized by GENEWIZ® (Suzhou, China). The sequences of sh-ASAP1 and sh-PLD1 were showed in Supplementary table 1. The pRNA-U6.1/Neo vector was used as control. Plasmid injection techniques: The plasmids were diluted with the normal saline. Mice were anaesthetized and held immobile. Then, the plasmids were rapidly injected into the retro-orbital sinus using a 5 mL injector needle.

Ethics statement

All animals involving in the experiments were complied with the guidelines of Declaration of Helsinki and approved by the Animal Care and Use Committee of Institute of Radiation Medicine, Chinese Academy Medical Sciences (Approval no. 2020–403).

Statistical analysis

Each experiment was repeated at least three times. Cumulative data are shown as mean ± SD with respect to the number of samples (n) in each group. Significance was assessed by comparing the mean values using Student’s t test and Wilcoxon rank sum test for independent groups as follows: *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed using GraphPad Prism. For additional information please see Supplemental Material.

Results

IVIg mitigates radiation-induced hematopoietic toxicity in a sex-specific fashion

As shown in Fig. 1A, male and female mice were treated with IVIg via intravenous injection 24 h before total body irradiation (TBI) and termed as IVIg(pre), the mice were treated with IVIg immediately after TBI and termed as IVIg(post). After 15 days, the male and female mice were euthanized, and the hematopoietic tissues were collected and analyzed. Intriguingly, both pre and post IVIg treatment restored radiation-atrophied spleen and thymus in female mice, but only IVIg(post) increased the weight of thymus in male mice (Fig. 1B-I). IVIg also improved the hemogram parameters in female mice, as judged by elevation of white blood cells (WBC) and lymphocyte proportion (LY%), reduction of the percentage of neutrophile granulocyte (NE%) in peripheral blood (PB) following TBI (Fig. 1J-L). For male mice, IVIg failed to ameliorate the aneretic hemogram parameters, only WBC experienced a slightly increase in mice from IVIg(post) group (Fig. 1M and supplementary Fig. 1A, B). Together, all the evidence indicates that IVIg might be an efficacious agent to fight against radiation-induced hematopoietic toxicity in female only, while the male counterparts do not respond significantly.

Fig. 1.

(A) Schematic illustrating male (green) and female (red) mice were treated with IVIg via intravenous injection before (IVIg (pre)) or after (IVIg (post)) total body irradiation (TBI). (B-E) Photographs (B, D) and weight (C, E) of dissected spleens from the four groups. (F-I) Photographs (F, H) and weight (G, I) of thymuses from the four groups. (J-M) White blood cell (WBC) counts (J, M), percentage of neutrophile granulocyte (NE%, K) and lymphocytes (LY%, L) in PB. Error bars represent means ± SD, n = 12 per group. Data representing two independent experiments were analyzed with unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.

IVIg fights against intestinal radiation toxicity in a sex-dependent manner

Owing to the better efficacy of IVIg(post) in mitigating radiation-induced hematopoietic injury, we chose this method throughout the subsequent experiments. Abdominal local irradiation curtailed the colons which were prolonged by IVIg injection in both male and female mice (Fig. 2A, B and supplementary Fig. 2A, B). ELISA assay showed that IVIg treatment only decreased the level of tumor necrosis factor-ɑ (TNFɑ) but not interleukin-1 (IL-1) and interleukin-6 (IL-6) in small intestine from male mice however, reduced the levels of the three proinflammatory factors in females (Fig. 2C-F and supplementary Fig. 2C, D), indicating that IVIg alleviates radiation enteritis overtly in female mice but slightly in males. Intravenous injection of IVIg stabled the structure of small intestinal villi, heightened the number of goblet cells in abdominal local irradiated female mice, but the therapeutics efficacy was not obvious in male counterparts (Fig. 2G, H). qRT-PCR assay further revealed that IVIg administration up-regulated the expression of epithelial integrity-related genes in female mice only (Fig. 2I, J and supplementary Fig. 2E), but not in males (Fig. 2K, L and supplementary Fig. 2F), indicating that IVIg only improves the radiation-damaged small intestinal integrity in female mice. In addition, IVIg also decreased the levels of Nrf2 and reactive oxygen species (ROS) in small intestine from irradiated female mice (Fig. 2M, N), and the changes in male mice were negligible (Fig. 2O, P). Together, IVIg strengthens intestinal barrier function and epithelial integrity, hinders radiation enteritis and reduces ROS levels in a sex-dependent fashion.

Fig. 2.

Male and female mice were exposed to 12 Gy TAI and the target tissues were obtained at day 21. (A, B) Photographs of colons from both sexes of mice. (C-F) The levels of small intestinal IL-6 and TNFɑ. (G, H) Representative H&E (scale bar, 200 μm) and PAS (scale bar, 40 μm) staining showed the morphology of small intestine per group. The arrows represented goblet cells. (I-L) The epithelial integrity related genes MDR1 and Glut1 of small intestine. (M−P) The expression levels of Nrf2 and the content of ROS in the small intestine were examined. Data representing two independent experiments were analyzed with unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Gut microbiota experiences a sex-specific alteration following IVIg injection

Our previous study reported that sexual dimorphism in gut microbiome dictated the curative efficacy of radiation toxicity. Thus, we analyzed the gut microbiota configurations in irradiated male and female mice with or without IVIg treatment. 16S rRNA sequencing showed that IVIg unchanged the α-diversity of gut microbes in fecal pellets from irradiated male and female mice (Fig. 3A, B and supplementary Fig. 3A-D). Notably, IVIg decreased the β-diversity of enteric microorganisms in irradiated male mice (Fig. 3C and supplementary Fig. 3E); however, the same remedy increased that in female counterparts (Fig. 3D and supplementary Fig. 3F). Unweighted and weighted principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) all exhibited more significant separation of gut microorganisms in irradiated female mice with IVIg injection compared with male counterparts (Fig. 3E-H and supplementary Fig. 3G, H). Although analysis of similarity (ANOSIM) showed an obviously difference between the gut microbial communities from both sexes with or without IVIg treatment (supplementary Fig. 3I, J), all the evidence indicated that IVIg induced more pronounced fluctuations of gut microbiota in female mice.

Fig. 3.

(A, B) Alpha diversity represented by the observed species number of intestinal bacteria from TAI mice with or without IVIg treatment. (C, D) Boxplots showed the inter-variation of TAI and IVIg group. (E-H) Unweighted unifrac-based PCoA plot and NMDS plot revealed the separation of bacterial community cluster between the irradiated mice with or without IVIg injection. Each circle represents a single sample. (I, J) The number of bacteria species and box plot of the weighted unifrac distances were compared between the irradiated male and female mice with IVIg injection. (K, L) Unweighted unifrac-based PCoA and NMDS showed the shift in gut bacteria between the irradiated male and female mice with IVIg injection. (M) ANOSIM determined the significant difference of microbiota configurations between the two sexes with IVIg injection. (N-P) 16S high-throughput sequencing identifies the most differential abundant strain bacteria between the two groups. Significant differences determined by Wilcoxon rank sum test, n = 12 per group.

Next, we compared the enteric bacteria taxonomic proportions between the irradiated male and female mice with IVIg injection. Although α- and β-diversity showed no difference (Fig. 3I, J and supplementary Fig. 3K-M), NMDS, unweighted and weight PCoA all represented visible separation of gut bacterial communities between the two cohorts (Fig. 3K, L and supplementary Fig. 3N). ANOSIM further validated the sex-specific gut microbiota configurations between male and female mice with the same treatment (Fig. 3M). In detail, the female mice harbored higher abundance of g_Roseburia_s_Lachnospiraceae_bacterium_DW52, and lower abundance of g_Enhydrobacter_s_Moraxella_osloensis and g_Blautia_s_Blautia_glucerasea compared with the male mice after the manipulation (Fig. 3N-P). Together, our observations demonstrate that IVIg injection structures a sex-specific gut microbiota configuration in abdominal local irradiated male and female mice.

Intestinal Lachnospiraceae potentiates the radioprotection of IVIg in male mice

Next, we scrutinized the variation trend of enteric Lachnospiraceae in male and female mice following abdominal local irradiation. Intriguingly, radiation stimuli reduced the relative abundance of Lachnospiraceae in male mice which was elevated in female counterparts (Fig. 4A, B). In light of all the obtained results above, we inferred that Lachnospiraceae might implicate in IVIg-mediated radioprotective effects in female mice. Thus, we cultured Lachnospiraceae in vitro and treated the irradiated male mice with this anaerobium via oral route combined with IVIg injection. As expected, Lachnospiraceae replenishment restored the atrophic hematogenic organs and raised the number of WBC in PB compared with IVIg treatment alone (Fig. 4C-F and supplementary Fig. 4A). In the abdominal local irradiation models, Lachnospiraceae combined with IVIg mitigated radiation enteritis, as judged by longer colons (Fig. 4G, H) and lower proinflammatory factor levels in small intestine (Fig. 4I, J and supplementary Fig. 4B). Immunohistochemical (IHC) staining showed that Lachnospiraceae administration restructured the intestinal villi and increased the goblet cells (Fig. 4K). Lachnospiraceae replenishment also improved the epithelial integrity and reduced ROS levels in abdominal local irradiated male mice combined with IVIg injection (Fig. 4L-N and supplementary Fig. 4C, D). Together, Lachnospiraceae synergizes IVIg-mediated radiation toxicity repairment in male mice.

Fig. 4.

(A, B) The relative abundances of g_Roseburia_s_Lachnospiraceae_bacterium _DW52 in both sexes with TAI based on 16S rRNA sequencing. Then, the irradiated male mice were injected with IVIg or IVIg combined with Lachnospiraceae. (C, D) Photographs of dissected spleen and thymus from the two groups. (E) The weight of dissected thymuses from irradiated male mice treated with IVIg alone or IVIg combined with Lachnospiracea. (F) WBC in PB were tested between the two groups. (G, H) Photographs and the length of colon from the two groups. (I, J) IL-6 and TNFɑ in the small intestine from the two group. (K) Representative H&E (scale bar: 100 μm) and PAS (scale bar: 20 μm) staining showed the morphology of small intestine per group. The arrows represented goblet cells. (L-N) The levels of Glut1, Nrf2 and ROS in the small intestine from the two group. Data representing two independent experiments were analyzed with unpaired Student’s t test: *P < 0.05, ***P < 0.001.

IVIg shapes the gut microbiota metabolome in a sex-specific manner

We also assessed the metabolome of gut microbiota in abdominal local irradiated male and female mice by LC-MS/MS analyses. As expected, the intestinal microorganism metabolome was quite diverse between irradiated male and female mice with IVIg injection (Fig. 5A, B). Volcano plot illustrated that the experimental male and female mice carried characteristic metabolites (Fig. 5C, D). Conjoint analysis between 16S rRNA sequencing and metabolome showed that hypoxanthine might be a potential metabolite produced by Lachnospiraceae (Fig. 5E). Higher frequency of enteric Lachnospiraceae was observed in irradiated female mice, heatmap based on LC-MS/MS analyses further revealed the higher level of hypoxanthine in the female mice compared with the males following abdominal irradiation (Fig. 5F). In addition, we examined the level of hypoxanthine in experimental male mice with Lachnospiraceae replenishment and obtained that Lachnospiraceae treatment heightened the levels of hypoxanthine in PB and small intestine tissues (Fig. 5G, H). Together, our observations indicate that IVIg molds distinct gut microbiota metabolite profiles in irradiated male and female mice, and hypoxanthine might be a metabolite derived from Lachnospiraceae.

Fig. 5.

The metabolome of gut microbiota in abdominal local irradiated male and female mice were assessed by LC-MS/MS analysis. (A, B) PCA plot revealed that the intestinal microorganism metabolome cluster was separately between male and female mice. (C, D) Volcano plots of different intestinal microorganism metabolite from abdominal local irradiated male and female mice with IVIg treatment. In the volcano plots, each point represented a metabolite. Red dots represented the up-regulated metabolites and green ones represented the down-regulated metabolites. (E) Correlation between bacterial phyla Lachnospiraceae and fecal metabolites. (F) Hierarchical cluster analysis for the metabolites in male and female mice with abdominal local irradiation. The arrow pointed to hypoxanthine. (G, H) The levels of hypoxanthine were tested in peripheral blood (G) and small intestine (H) from IVIg injected male mice with Lachnospiraceae replenishment or not. Data representing two independent experiments were analyzed with unpaired Student’s t test: *P < 0.05, ***P < 0.001, n = 10 per group.

Hypoxanthine combined with IVIg fights against radiation toxicity in male mice

To determine the synergistic effects of hypoxanthine, the irradiated male mice were treated with hypoxanthine via oral route combined with IVIg intravenous injection. ELISA assays showed that the hypoxanthine was accumulative in small intestine but not in PB (Fig. 6A, B). In abdominal local irradiation male models, hypoxanthine replenishment prolonged the colon length (Fig. 6C, D) and lessened the levels of IL-1, IL-6 and TNFɑ in small intestine tissues (Fig. 6E, F and supplementary Fig. 4E). In parallel with Lachnospiraceae, hypoxanthine addition rehabilitated intestinal villi, increased the number of goblet cells, up-regulated the epithelial integrity-related genes and reduced the ROS level in small intestine (Fig. 6G-K and supplementary Fig. 4F). In total body irradiation models, oral gavage of hypoxanthine failed to restore the spleen and thymus tissues maybe because the hypoxanthine was unavailable in circulatory system (supplementary Fig. 4G, H). Thus, the male mice were treated with hypoxanthine by intraperitoneal injection combined with IVIg and exposed to total body irradiation. Intriguingly, hypoxanthine was cumulative in PB (Fig. 6L), resulting in ameliorating atrophic spleen and thymus, and increasing number of WBCs (Fig. 6M-P and supplementary Fig. 4I). We also assessed the radioprotection of Lachnospiraceae or hypoxanthine without IVIg. The results showed that oral gavage of Lachnospiraceae or hypoxanthine accumulated hypoxanthine in small intestine tissues, but failed to protect against intestinal radiation injuries without IVIg combination in male mice (supplementary Fig. 5). Together, our observations demonstrate that Lachnospiraceae-derived hypoxanthine potentiates the radioprotective effects of IVIg in male mice.

Fig. 6.

(A, B) The levels of hypoxanthine in small intestine and peripheral blood from IVIg injected male mice with or without hypoxanthine replenishment were examined. (C, D) Photographs and length of dissected colon from the two groups. (E, F) The levels of small intestinal IL-6 and TNFɑ. (G) Representative H&E (scale bar: 100 μm) and PAS (scale bar: 20 μm) staining showed the morphology of small intestine per group. The arrows represented goblet cells. (H-K) The levels of Glut1, MDR1, Nrf2 and ROS in the small intestine from the two groups. (L) The level of hypoxanthine was examined in PB from IVIg treated male mice via intraperitoneal injection with or without hypoxanthine replenishment. (M, N) Photographs of dissected spleen and thymuses from the two groups. (O) The weight of dissected thymuses from irradiated male mice with hypoxanthine replenishment via intravenous injection. (P) WBC counts in PB was measured from the two groups. Data representing two independent experiments were analyzed with unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001, n = 10 per group.

IVIg protects against radiation toxicity via PLD1

Finally, we investigated the radioprotective mechanism of IVIg. Transcriptome analysis of small intestine tissues showed that the Fc γ receptor-mediated phagocytosis pathway was activated in female mice but not in males following abdominal local irradiation (supplementary Fig. 6a). Given the Fc region of IVIg is the critical fragment for the initiation of responses, we screened the genes related to Fc γ receptor-mediated phagocytosis pathway and collected two genes which were up-regulated in female mice but down-regulated in males based on transcript sequencing (supplementary Fig. 6B). Then, we validated the expression of the two genes, phospholipase D1 (PLD1) and ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1), with an expanded sample size. qRT-PCR assay revealed that abdominal local irradiation indeed down-regulated the expression of PLD1 and ASAP1 in small intestine from male mice, but up-regulated those in females (Fig. 7A, B). We also found that Lachnospiraceae or hypoxanthine administration via oral route only elevated the level of small intestinal PLD1 but not ASAP1 in abdominal irradiated male mice with or without IVIg injection (Fig. 7C, D and supplementary Fig. 6C, D), suggesting that PLD1 might be the key element in the radioprotection of IVIg. Next, we silenced intestinal PLD1 and ASAP1 respectively by hydrodynamic-based gene delivery assay in abdominal irradiated female mice with IVIg injection. qRT-PCR validated the reduction of PLD1 and ASAP1 expression in the small intestine (Fig. 7E, F). Importantly, silencing PLD1 not ASAP1 erased the protection of IVIg to radiation enteritis, as judged by curtailing colon length (Fig. 7G, H) and elevating proinflammatory cytokines (Fig. 7I, J and supplementary Fig. 6E). PLD1 knockdown also rendered sparser intestinal villi and fewer goblet cells, destructed epithelial integrity and heightened ROS level in small intestine, which were negligible in the female mice with ASAP1 silencing (Fig. 7K-O and supplementary Fig. 6F). Together, our observations demonstrate that PLD1 plays a vital role in IVIg-mediated radioprotection in male and female mice.

Fig. 7.

(A, B) The expression of PLD1 and ASAP1 in small intestine. (C, D) The expression of PLD1 and ASAP1 in small intestine from male mice in IVIg group, IVIg combined with Lachnospiraceae group and IVIg with hypoxanthine replenishment group. (E, F) The expression levels of PLD1 and ASAP1 in small intestine from irradiated female mice with silencing intestinal PLD1 or ASAP1 respectively by hydrodynamic-based gene delivery assay. (G, H) Photographs and the length of colons from irradiated female mice with sh-PLD1 or sh-ASAP1 injection. (I, J) IL-6 and TNFɑ in the small intstine from IVIg group, IVIg with sh-PLD1 group and IVIg with sh-ASAP1 group. (K) Representative H&E (scale bar: 100 μm) and PAS (scale bar: 20 μm) staining showed the morphology of small intestine per group. The arrows represented goblet cells. (L-O) The levels of Glut1, MDR1, Nrf2 and ROS in small intestine from the three group. Data representing two independent experiments were analyzed with unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001, n = 10 per group.

Discussion

Hematopoietic system and alimentary canal related toxicity rank as the top 2 complications entwined with systemic therapy and radiotherapy. Both chemotherapy and radiotherapy drive varying degrees of bone marrow toxicity, with myelosuppression, neutropenia and myeloid skewing as clinical manifestations. These side effects result in hypoimmunity of patients and halt the remedies prematurely. Radiotherapy for pelvic and abdominal malignancies causes radiation intestinal toxicity [26] as judged by intestinal obstruction, gastrointestinal tract hemorrhage, enteritis and colitis etc. The complications degrade the life quality of patients and starve for therapeutic strategies in clinical application. To date, amifostine is the only drug approved by the FDA for the rehabilitation of cancer patients with radiotherapy; however, amifostine is potential to radio-protect the tumor and negatively impact tumor control and patient survival. IVIg is administered for various indications and considered as a safe agent generally. We found that intravenous injection of IVIg mitigated radiation-induced hematopoietic and GI tract injuries in female mice only, but not in males. It suggests that IVIg is a potential radioprotectorant fighting against radiation toxicity in a sex-dependent manner. IVIg is manufactured from large pools of donor plasma, the preparation contains substantial proportion of primary IgG from both sexes. Thus, the sexual dimorphism of therapeutics efficacy is independent of IVIg components. The traditional notion that antibody neutralizing toxin through Fab region-antigen mutual interactions has relied on in vitro assays, which ignore the sophisticated microenvironments in vivo conditions. Recent evidence underpins that the Fc fragment-Fc γ receptor interactions are crucial for facilitating optimal in vivo protection [27], [28]. Intriguingly, radiation exposure alters Fc γ receptor-related signaling in vivo. We analyzed the transcriptome of small intestine from local irradiated mice of both sexes and observed that Fc γ receptor-mediated phagocytosis showed different phenotypes. Compared with male mice, the signaling pathway was activated and the relative genes were up-regulated in female counterparts. In this respect, the effectiveness of IVIg in females might attribute to the activation of Fc γ receptor-mediated signaling pathway. We screened and focused on two genes which were up-regulated in irradiated female mice but down-regulated in males. Silencing PLD1 in female mice overtly erased the radioprotection of IVIg. PLD1 is the major source of signal-activated phosphatidic acid generation downstream of G protein-coupled receptors, receptor tyrosine kinases and integrins etc [29]. Thus, it suggests that IVIg fights against hematopoietic and intestinal radiation toxicity at least partly dependent on Fc γ receptor-mediated phagocytosis, and the different responses of Fc γ receptor-mediated signaling to radiation stimuli in male and female induce the sex-specific therapeutic efficacy of IVIg. Clinically, the usage of IVIg can be classified into 2 categories simply, i.e. as a replacement therapy in immunodeficiencies and as immunomodulatory or anti-inflammatory therapy in other diseases. In the present study, we also observed slightly mitigation of radiation-induced inflammation such as longer colon and lower TNF-α level in male mice, which might depend on the anti-inflammatory effects of IVIg. In addition, immunotherapy offers an alternative to conventional cancer therapy, and the combination of immunotherapy and radiotherapy facilitates the therapeutic efficacy [30], [31]. As a classic immunomodulator, IVIg has a potential to play synergistic roles with radiotherapy or immunotherapy theoretically, which deserves further study.

Sexual dimorphism is common occurrence in mammals and visible in almost all aspects of individuals, from brain structure to gut microbiome configuration [32], [33]. Sex influences the body clock and confers temporal paradigm on behavior and physiology to align homeostatic responses with anticipated and intricate changes in the milieu [34]. Sex disparities in diagnosis and therapy are encountered frequently in clinical conditions which has raised concern in epidemiology, pathophysiology, clinical manifestations, disease progression, and response to treatment [35]. For example, men and women represent dimorphism in substrate supply and utilization, excess lipids deposition and stored lipids mobilization in metabolic organs, resulting in different cardiometabolic risk factors and suffering from sex-specific types of cardiometabolic disorders [36], [37]. Our previous study identified that male and female mice harbored sex-specific gut microbiota, the changes in the gut microbiota also revealed a sex-dependent manner after radiation exposure, [19] importantly, they showed different responses to the same treatment for radiation toxicity [25]. Sexual dimorphism in gut microbiome influences the occurrence and development of diseases, such as type Ⅰ diabetes mellitus, non-alcoholic fatty liver disease and coronary artery disease, in a sex-specific way [38], [39], [40], [41]. In line with the evidence, we also found that the sexual disparities in gut microbiota dictated the therapeutics efficacy of radiation toxicity in this study. Bacteria, archaea, viruses and fungi dwelt in digestive tract communicate with each other, forming a sophisticated and dynamic microecosystem with the host. To clarify the vision of sex bias in pathological and pharmacological manifestations and establish comprehensive prevention and curation strategies for both sexes, we teased apart the data from 16S rRNA sequencing. Abdominal local irradiated shaped the gut microbiota in a sex-specific way. Intriguingly, a strain belongs to Lachnospiraceae, g_Roseburia_s_Lachnospiraceae_bacterium_DW52, showed an incremental trend in female mice but an inverse trend in males following irradiation. These contrary alterations might rely on the differences in genetic characteristics, hormone profile and gut microbiome between male and female mice. Transplantation of female fecal microbiota to irradiated males was able to mitigate the radiation-induced toxicity. In addition, Guo and colleagues report a Lachnospiraceae pool protects the hosts against radiation challenge [42]. However, we observed that an individual strain in the family Lachnospiraceae alone could not protect male mice against radiation injuries, validating the systemic complexity of gut microbiota in radioprotection. However, replenishment of Lachnospiraceae combined with IVIg ameliorated radiation-induced hematopoietic and GI tract toxicity overtly in males, suggesting Lachnospiraceae might be a bio-synergist for IVIg to mitigate radiation toxicity efficaciously. The gut microbiota interacts with the host through small molecules which are produced as intermediate or end products of microbial metabolism [43]. Germ-free and conventional mice carry different metabolite profiles in plasma, urine, liver, kidney and gut tissues, inferring the importance of microbial metabolites in mammalian biology [14]. Metabolomics analysis pointed out that hypoxanthine might be a potential metabolite of Lachnospiraceae. Oral gavage of Lachnospiraceae indeed elevated the levels of hypoxanthine in small intestine tissues and serum. Hypoxanthine is conductive to epithelium function through enhancing cellular energetics and cytoskeletal capability and serves as a biomarker of barrier function [44]. Same as Lachnospiraceae, oral gavage of hypoxanthine improved the radioprotection of IVIg in male mice as well. For male mice, abdominal local irradiation down-regulated PLD1 in small intestine, resulting in neutralizing the radioprotective effects of IVIg. Thus, we assessed the expression of small intestinal PLD1 in male mice with Lachnospiraceae or hypoxanthine treatment. Intriguingly, oral gavage of Lachnospiraceae or hypoxanthine up-regulated the PLD1 expression in males with or without IVIg injection. PLD1 has been reported to mitigate tissue injuries, such as muscles, kidneys and lungs [45], [46], [47]. Here, our findings bolster that PLD1 also plays an important role in ameliorating radiation intestinal injury. Replenishment of hypoxanthine via oral route failed to mitigate bone marrow toxicity, probably because the unavailability of hypoxanthine in PB following oral gavage. Both oral gavage of Lachnospiraceae and intraperitoneal injection of hypoxanthine enriched hypoxanthine in PB and potentiated the radioprotection of IVIg to hematopoietic injury. The results further bolster that hypoxanthine is a key mediator for IVIg to improve radiation toxicity. Considering the evidence from experimental female mice, we conclude that the radioprotection of IVIg might be dependent on Lachnospiraceae/hypoxanthine/PLD1 axis. However, the underlying mechanism by which hypoxanthine increases PLD1 expression required further study.

Conclusions

Our data demonstrates that IVIg fights against radiation toxicity in a sex-specific, gut microbiome-dependent way. Radiation exposure elevates the abundance of intestinal Lachnospiraceae and the expression of PLD1 in small intestine tissues in females but reduces those in males. Thus, IVIg injection protects females against radiation toxicity. For males, replenishment of Lachnospiraceae or hypoxanthine, a metabolite derived from Lachnospiraceae, dictates the radioprotective function of IVIg. Together, our findings bolster that sexual disparities in gut microbiome impacts the therapeutics options for radiation injuries, and shed light on the precision rehabilitation based on the sex of tumor patients.

Compliance with Ethics Requirements

All Institutional and National Guidelines for the care and use of animals (fisheries) were followed.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: