Abstract

   T-2 toxin, a fungal secondary metabolite produced by toxigenic Fusarium
   species, poses a significant threat to grain food and feed due to its
   potential to cause intestinal inflammation in livestock and poultry.
   Macrophages play a crucial role as integral components of the body's
   immune system during intestinal inflammation. This study aimed to
   elucidate the mechanism behind the inflammatory response triggered by
   T-2 toxin in macrophages. Compared to the control group, gavage
   administration of T-2 toxin (0.33, 1, and 4 mg kg^−1) led to a decrease
   in body weight and feed intake, along with histopathological
   alterations in the colon of mice. In addition, T-2 toxin induced the
   upregulation of macrophage-derived cytokines like IL-1β, IL-6, and
   TNF-α, as well as a rise in the population of F4/80^+ macrophages in
   the colon. T-2 toxin also led to the upregulation of IL-1β, IL-6, and
   TNF-α in mouse bone marrow-derived macrophages (BMDMs). Furthermore,
   the transcriptomic analysis of BMDMs exposed to T-2 toxin (10 nM)
   identified the "TNF signaling pathway," "Lipid and atherosclerosis,"
   "Epstein-Barr virus infection," "MAPK signaling pathway," and the
   "NF-kappa B signaling pathway" as the top five significantly enriched
   pathways. Subsequently, twelve inflammation-related genes were randomly
   chosen for validation through quantitative reverse transcription PCR
   (RT-qPCR), with the results corroborating those from the transcriptomic
   analysis. The comprehensive analysis of transcriptome data highlights
   the activation of several signaling pathways associated with the
   inflammatory response following T-2 toxin-induced BMDMs, offering
   potential therapeutic targets for the prevention and treatment of T-2
   toxin-induced intestinal inflammation.

   Keywords: T-2 toxin, Intestinal inflammation, Bone marrow-derived
   macrophages, Transcriptomic analysis

Graphical abstract

   [33]Image 1
   [34]Open in a new tab

Highlights

     * •
       T-2 toxin induced macrophage infiltration and intestinal
       inflammation in mice.
     * •
       T-2 toxin induced inflammatory response in mouse bone
       marrow-derived macrophages.
     * •
       T-2 toxin induced strong inflammatory transcriptomic changes in
       mouse macrophages.

1. Introduction

   T-2 toxin, a fungal secondary metabolite produced by various toxigenic
   Fusarium species, has been extensively investigated due to its early
   discovery, high toxicity, and wide presence in food and animal feed
   [[35]1]. Its potent toxicity leads to intestinal inflammation, posing
   significant threats to human health and animal husbandry [[36]2].
   Considering the immune system as a major target of T-2 toxin toxicity,
   considerable attention has been given to studying its impact on immune
   responses in recent years.

   T-2 toxin can distribute throughout the body and reach various organs
   without the need for transporters or adjuvants [[37]3]. The toxin
   undergoes rapid absorption in the gastrointestinal (GI) tract [[38]4],
   leading to damage in nearly all GI tract cells and influencing nutrient
   absorption even at minimal toxin concentrations [[39]5]. Obremski et
   al. found the impact of T-2 toxin on the percentages of CD4^+ and CD8^+
   T lymphocytes, CD21^+ B cells, as well as the mRNA expression levels of
   related cytokines in porcine ileum [[40]6].

   Macrophages are an important part of the body's defense against
   infection and disease, playing a pivotal role in the immune system
   [[41]7,[42]8]. They are distributed throughout various tissues,
   including in the lamina propria of the gut. Most adult intestinal
   macrophages originate from the monocytic cell lineage that emigrates
   from the bone marrow (BM) and play a crucial role in maintaining
   intestinal homeostasis. Noel et al. established a co-culture model of
   the gut and macrophages, demonstrating that the depletion of
   macrophages adversely affects the subsequent differentiation of
   intestinal epithelial cells [[43]9]. Sehgal A et al. demonstrated that
   colony-stimulating factor 1 (CSF1)-dependent macrophages within the gut
   wall are capable of repairing enteritis induced by inflammation or
   chemotherapy and are essential for sustaining the small intestinal stem
   cell niche [[44]10]. In inflammatory conditions, circulating monocytes
   originating from the BM infiltrate the inflamed intestinal tissue and
   differentiate into macrophages [[45]11]. Research by Nanthakumar
   indicated that neonatal colitis is characterized by a high rate of
   macrophage infiltration in the inflamed regions, underlying the role of
   macrophages as the first line of defense [[46]12]. Additionally,
   Swirski et al. revealed that the spleen serves as a critical reservoir
   for monocytes in mice, exhibiting a phenotype similar to that of
   blood-derived monocytes [[47]13]. These findings suggest that the
   spleen serves as an additional primary source of monocytes, which
   differentiate into macrophages and facilitate their migration to the
   inflamed intestinal tissue.

   Mycotoxins have the potential to influence the activity and function of
   macrophages. For example, elevated levels of gibberellins can induce
   apoptosis in macrophages, consequently diminishing their ability to
   release inflammatory cytokines [[48]14]. Trichothecene mycotoxins,
   among the naturally occurring food contaminants identified as the most
   dangerous [[49]15], profoundly impact the immunomodulatory function of
   macrophages. Even low concentrations of T-2 toxin disrupt the
   recognition of pathogen-associated molecular patterns by porcine
   primary macrophages, resulting in reduced Toll-like receptor expression
   and impairing the immune system [[50]16]. Further research is warranted
   to elucidate the molecular mechanisms responsible for the
   immunotoxicity of T-2 toxin in inducing intestinal inflammation in
   macrophages. Transcriptome sequencing also called as RNA sequencing or
   RNA-Seq is a high-throughput sequencing technology that enables the
   characterization of all RNA transcripts present in a given sample at a
   specific moment [[51]17].

   In this study, we assessed intestinal inflammation in mice treated with
   T-2 toxin. Given the crucial role of macrophages in maintaining
   intestinal homeostasis and medicating the immune response, we evaluated
   macrophage infiltration in the intestines of mice and the inflammatory
   response in mouse bone marrow-derived macrophages (BMDMs).
   Additionally, we employed RNA-seq technology to sequence the
   transcriptome of BMDMs in response to T-2 toxin-induced inflammation,
   aiming to gain further insights into the regulatory mechanisms and
   molecular pathways underlying macrophage function affected by T-2
   toxin.

2. Materials and method

2.1. T-2 toxin solution preparation

   T-2 toxin (CAS number: 21259-20-1, 99.0 % purity) was purchased from
   Qingdao Pribolab Biotechnology Company (Qingdao, China). The T-2 toxin
   was dissolved in dimethyl sulfoxide (DMSO, CAS number: 67-68-5, 99.0 %
   purity, Sangong Bioengineering, Shanghai, China) to create a solution
   with a concentration of 25 mg mL^−1, and stored at −20 °C, protected
   from light.

2.2. Mice, treatments, and samples collection

   SPF 8-10-weeks-old C57BL/6 male mice were purchased from Hangzhou
   Qizhen Laboratory Animal Science and Technology Co., LTD. All mice were
   maintained in pathogen-free conditions with standard laboratory chow
   and water ad libitum. The protocol was approved by the Committee on the
   Ethics of Animal Experiments of Zhejiang Academy of Agricultural
   Science. Project Proposal Number 2023ZAASLA020. After one week of
   acclimatization, the mice were randomly divided into 4 groups, with 5
   mice in each group. The mice received gavage administration of normal
   saline (Control), or T-2 toxin at a dose of 0.33, 1, or 4 mg kg^−1,
   respectively. Body weight and food intake were monitored daily starting
   from the initial day of gavage. On day 7, spleen and colon tissues were
   collected from mice for subsequent Hematoxylin-Eosin (H&E) staining or
   RT-qPCR analysis.

2.3. H&E staining

   Colon tissues were fixed in a 4 % paraformaldehyde solution for 24 h,
   dehydrated using a HistoCore PEARL tissue processor (Leica Biosystems,
   Nussloch, Germany), and subsequently embedded in a paraffin block using
   a HistoCore Arcadia paraffin embedding machine (Leica Biosystems,
   Nussloch, Germany). Tissue sections with a thickness of 5 μm were
   stained with H&E to observe colonic inflammation and histopathological
   damage. Staining was carried out as per the manufacturer's protocol
   using an H&E staining kit (Golden Clone Biotechnology, Beijing, China).

2.4. Immunofluorescence staining

   Colon tissue sections were deparaffinized using xylene and a graded
   series of ethanol. Antigen retrieval was performed by immersing the
   sections in a 10 mmol/L EDTA solution (pH 8.0) and heating them in a
   water bath at 95 °C for 20 min. Subsequently, the sections were rinsed
   with phosphate-buffered saline (PBS, pH 7.2) and incubated with a
   blocking buffer (1 % BSA/0.3 % Triton X-100/1 % normal goat serum in
   PBS) at room temperature for 30 min. The sections were then incubated
   with anti-mouse F4/80 primary antibody (Invitrogen, Carlsbad, USA) at
   4 °C overnight. After washing with PBS, the sections were incubated
   with Alexa Fluor 488-conjugated (IgG) secondary antibody (Invitrogen,
   Carlsbad, USA) for 1 h at room temperature. Finally, the sections were
   incubated with hoechst 33342 at room temperature for 5 min.
   Fluorescence images were captured using a confocal laser scanning
   microscope (Zeiss, LSM 900, Oberkochen, Germany). The F4/80-positive
   cells were quantified using ImageJ software (National Institutes of
   Health, USA).

2.5. Cell culture and treatment

   Bone marrow-derived macrophages (BMDMs) were obtained from the tibia
   and femoral bone marrow of C57BL/6 male mice, and maintained in
   Iscove's Modified Dulbecco's Medium (IMDM, Thermo Fisher Scientific,
   Grand Island, USA) supplemented with 10 % (vol/vol) fetal bovine serum
   (FBS, Thermo Fisher Scientific), l-glutamine, sodium pyruvate, 100
   U mL^−1 penicillin-streptomycin, and 20 ng mL^−1 macrophage
   colony-stimulating factor (M-CSF, R&D Systems, Minneapolis, USA) for 7
   days. For experiments, BMDMs were seeded into plates containing IMDM
   supplemented with 1 % FBS and incubated overnight. Subsequently, the
   BMDMs were exposed to T-2 toxin for 6 h for further RT-qPCR analysis
   and RNA-seq analysis.

2.6. cDNA synthesis and RT-qPCR analysis

   Total RNA was extracted from mouse tissues or BMDMs using TriPure
   Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) following
   the manufacturer's instructions. cDNA synthesis was performed using the
   PrimeScript RT reagent Kit (Takara, Japan) for reverse transcription.
   Real-time PCR was conducted using the iTaq™ Universal SYBR® Green kit
   (Bio-Rad, USA) on a Real-Time PCR detection system (Bio-Rad, CFX96,
   USA). The primer sequences of IL-1β, IL-6, TNF-α, and the internal
   reference gene β-actin are provided in [52]Supplementary Table 1. The
   relative expression level of target genes to β-actin was calculated
   using the 2^−ΔΔCt method and normalized to the control group.

2.7. RNA-seq bioinformatics analysis and RT-qPCR verification

   Total RNA was extracted from mouse BMDMs using TriPure Isolation
   Reagent, and mRNA with poly tails was enriched with magnetic beads with
   an Oligo (dT). The enriched mRNA was further purified, reverse
   transcribed, and synthesized into double-stranded cDNA. Subsequently, a
   library was constructed and subjected to quality control undergoing
   paired-end sequencing (PE150). The initial step involved performing
   data quality control on the raw data obtained from the sequencing
   machine. Afterward, the raw data underwent de-junctioning and
   low-quality filtering. The filtered data was then compared and aligned
   to the reference genome sequence using HISAT2 software [[53]18],
   resulting in the generation of a comparison reference genome.
   Transcript reconstruction was carried out using Stringtie software
   [[54]19] to accurately assemble transcripts and quantify the expression
   levels of each gene or transcript. Finally, differential expression
   analysis was conducted using DESeq2 [[55]20] and relevant pathways were
   discovered by enrichment analysis.

   By analyzing the transcriptome sequencing data with KEGG pathway
   analysis, 12 immunity-related differentially expressed genes (DEGs)
   were randomly selected and the results were verified using RT-qPCR. The
   primer sequences of the 12 genes are provided in [56]Supplementary
   Table 2.

2.8. Statistical analysis

   Data analysis was performed using GraphPad Prism 9 software. Data are
   represented as mean ± SEM. To examine the statistical differences in
   the quantitative analysis of the intensity of F4/80 in the sections of
   colons between control and T-2 toxin treatment group, an unpaired
   two-tailed Student's t-test was conducted. The other statistical
   differences between groups were assessed using a Tukey post-hoc test,
   one-way analysis of variance (ANOVA). A p value less than 0.05 was
   considered statistically significant.

3. Results

3.1. T-2 toxin induces intestinal inflammation in mice

   To investigate the impact of T-2 toxin on intestinal inflammation in
   mice, we administered varying doses of T-2 toxin (0.33, 1, and
   4 mg kg^−1) orally to the mice for 7 consecutive days. The low dose of
   0.33 mg kg^−1 T-2 toxin significantly reduced the body weight and food
   intake of mice. Similarly, increasing doses of T-2 toxin at 1 and
   4 mg kg^−1 led to a corresponding decrease in body weight and food
   intake in mice ([57]Fig. 1A and B). On day 7, the experiment concluded
   with histological examination of the mice's colon tissues. Mice treated
   with 0.33 mg kg^−1 T-2 toxin exhibited observable inflammatory cell
   infiltration in the colon, whereas those treated with 1 mg kg^−1 T-2
   toxin displayed a more pronounced inflammatory cell infiltration
   compared to the control group. Remarkably, the group treated with
   4 mg kg^−1 T-2 toxin experienced a loss of the colon's histological
   structure, with inflammatory cells infiltrating the mucosa and
   submucosa ([58]Fig. 1C). The expression levels of the inflammatory
   cytokines IL-1β, IL-6, and TNF-α were significantly upregulated in both
   splenocytes and colon tissues in a dose-dependent manner due to T-2
   toxin exposure ([59]Fig. 1D and E). These findings indicate that T-2
   toxin induces significant intestinal inflammation in mice and
   stimulates the expression levels of inflammatory cytokines.

Fig. 1.

   [60]Fig. 1
   [61]Open in a new tab

   The impact of T-2 toxin on intestinal inflammation in mice. The mice
   received oral administration of normal saline (Control), or T-2 toxin
   (0.33, 1, or 4 mg kg^−1), and body weight (A) and food intake (B) were
   monitored daily for 7 days. Data are mean ± SEM (n = 5). ∗∗p < 0.01,
   ∗∗∗p < 0.001 (one-way ANOVA test). (C) H&E staining of paraffin
   sections of colons isolated from Control or T-2 toxin-treated mice on
   day 7. Scale bar, 100 μm. RT-qPCR analysis of IL-1β, IL-6, and TNF-α
   expression in splenocytes (D) and colon (E) isolated from Control or
   T-2 toxin-treated mice on day 7. Data are mean ± SEM (n = 5).
   ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus Control (one-way ANOVA
   test).

3.2. T-2 toxin induces macrophages infiltration in the colon

   Since T-2 toxin induced inflammatory cell infiltration and upregulation
   of macrophage-derived cytokines in the colon, we employed
   immunofluorescence staining to detect the infiltration of F4/80^+
   mature macrophages in mouse colon tissues. As depicted in [62]Fig. 2A,
   there was almost no presence F4/80^+ macrophages in the colon sections
   of the control group. Conversely, mice treated with 1 mg kg^−1 T-2
   toxin displayed a markedly observable infiltration of macrophages in
   the colon. Quantitative analysis of the results revealed a significant
   increase in F4/80-positive cells within the T-2 toxin treatment group
   ([63]Fig. 2B).

Fig. 2.

   [64]Fig. 2
   [65]Open in a new tab

   The impact of T-2 toxin on macrophage infiltration in the colon. The
   mice received oral administration of normal saline (Control) or T-2
   toxin (1 mg kg^−1) for 7 days. (A) Detection of F4/80 protein
   expression with immunofluorescence staining in the sections of colons
   of mice. The nuclei were stained with hoechst 33342 (blue) and F4/80
   protein was labeled with Alexa Fluor 488 (green). Scale bar, 100 μm.
   (B) Quantitative analysis of the F4/80-positive cells in the sections
   of colons. Data are mean ± SEM (n = 5). ∗∗p < 0.01 versus Control
   (unpaired two-tailed Student's t-test). (For interpretation of the
   references to color in this figure legend, the reader is referred to