Soil causes gut microbiota to flourish and total serum IgE levels to decrease in mice

Traditional farm environments induce protection from allergic diseases. In this study, farm environmental factors were classified into three categories, environmental microbes, soil, and organic matter. To explore the impact of soil and environmental microorganisms on gut microbiota and immune function, mice were fed sterilized soil, soil microbes (in lieu of environmental microbes), or non-sterilized soil. Metagenomic sequencing results showed the intake of sterile soil i.e. inhaling a small amount of soil microbes in the air increased gut microbial diversity and the abundance of type III secretion system (T3SS) genes, and decreased serum immune globulin E (IgE) levels induced by 2-4-dinitrofluorobenzene(DNFB). The intake of soil microbes increased the abundance of genes involved in the metabolism of short chain fatty acids and amino acid biosynthesis. Meanwhile, the intake of soil increased gut microbial diversity, the abundance of T3SS genes and related infectious elements, and genes associated with the metabolism of short chain fatty acids and amino acid biosynthesis, and decreased serum IgE levels. Therefore, soil may be useful as a potential “prebiotic” promoting the reproduction and growth of some intestinal microorganisms that harbor bacterial secretion system genes, especially those of T3SS, whose abundance was positively and significantly correlated with innate immune function of mice.

biosynthesis of amino acids. The intake of soil, which included the components of both the 2 3 5 sterilized soil and the soil microbes, not only increased abundance in the intestinal microbiota of 2 3 6 T3SS genes and the related infectious elements, but it also significantly increased the abundance 2 3 7 of genes related to the metabolism of short-chain fatty acids and biosynthesis of amino acids. The  significantly lower than those observed in the mice that ingested soil or sterilized soil. Therefore, 2 4 5 soil may function as a "prebiotic" for various microbial strains or may be necessary, establishing a human intestine (Lennon and Jones, 2011).
(2) As a multi-pore structure, soil might provide the bactericides secreted by the human body such as antimicrobial peptides or IgA. conditions in which the mice were inoculated with microorganisms via the air. Our previous study 2 5 5 found that in a SPF animal facility, adding sterilized soil to mouse bedding changed the 2 5 6 composition of intestinal microflora of mice, but did not increase the diversity of the intestinal 2 5 7 microorganisms (Zhou et al., 2018). In the current study, the experiment was designed so the SS 2 5 8 group of mice were reared next to the MW mice, which consumed soil microbes via their water.

5 9
The open mouse cages could not prevent soil microbes from spreading to the SS mice. The results 2 6 0 demonstrated increased gut microbial diversity of the SS mice. In the current study, there was no significant difference in the immune function of mice fed 2 6 2 soil microbes compared with that of the Control mice. Further, the intake of soil microbes 2 6 3 significantly increased the abundance of genes involved in short-chain fatty acid metabolism and 2 6 4 amino acid biosynthesis. Short-chain fatty acids play a role in improving human immunity  During ingesting soil microbes, many bacteria pass through the gut, which theoretically should 2 6 8 stimulate the immune system of the mice and increase their immune function. There may be two reasons to explain our current results: (1) the Control group and MW group were raised in the 2 7 0 same SPF animal facility. The mouse cage cover was a reticular structure and the soil microbes Pathogenic microbes and T3SS genes were detected in both the SS group and Soil group of 2 7 5 mice, and the flagellar assembly gene was abundant in the Soil mice (Table S9 and Fig. S6).

7 6
However, no infection was detected in the blood of any animals tested, which may have been due 2 7 7 to these reasons: (1) the infection occurred during the early stage of the experiment and had resolved by the time we tested the blood of the mice; (2)  The current results also showed a strong correlation between IgE levels and the abundance of  mechanism of T3SS has been widely reported(Büttner, 2012), but the mechanism involved in 2 9 0 improving host immune function needs further study. In this study, a mouse model was used as the research approach, but there are great 2 9 2 differences in lifestyles and evolutionary relationships between humans and mice. Therefore, the 2 9 3 impact of soil on human intestinal microflora may differ and needs more experimental proof. In conclusion, our results showed that an important reason farm environments have protective 2 9 5 effects on allergic diseases is that soil can be used as a "prebiotic" to increase the diversity and harboring the genes of bacterial secretion system. Further mechanistic studies revealed that soil improved the natural immune function of mice mainly by increasing the abundance of genes of 2 9 9 bacterial secretion system of gut microbiota, especially those of T3SS. At total of 60 mice aged 3-4 weeks were randomly divided into four groups (n = 20/group). The 3 0 3 temperature of SPF animal facility was maintained at 24 ± 2 ℃ , humidity was 40 ± 5%, and the 3 0 4 lights were on a 12 h/12 h light/dark cycle. Bedding material were change once a week. Starting at 3 0 5 7 weeks of age, the SS group was fed a diet containing 5% sterilized soil (Fig. S1), the Soil group 3 0 6 was provided a diet containing 5% non-sterilized soil, and the MW group was provided drinking 3 0 7 water containing ~10 11 soil microbes. No treatment was performed for the Control group, which 3 0 8 continued to receive a standard lab diet and normal drinking water. After 42 d of treatment, feces from 10 mice in each group was randomly collected and stored and evaluated for serum IgE levels.

1 3
Soil was collected from farm ground at a depth greater than 0.5 cm, but no more than 10 cm.

1 4
The soil composition was analyzed using a Wavelength Dispersive X-Ray Fluorescence  Table S3. The soil was 3 1 6 sterilized using autoclave at 121 °C for 30 min, which was repeated three times at a 24 h interval.

1 7
Before use, the sterilized soil and non-sterilized soil were crushed and mixed with the mouse diet.

1 8
Sterilized or non-sterilized soil feed was stored at −20 °C.

1 9
Soil microbes were isolated as following: Fresh farm soil was collected and mixed with sterile 3 2 0 water at 2:1 (w/v). After stirring with a magnetic rod for 20 min, the solution was allowed to stand 3 2 1 undisturbed for 10 min. The supernatant was then collected and centrifuged at 41 × g for 5 min.

2 2
The supernatant was collected and allowed to stand for 48 h. The supernatant was again collected 3 2 3 and centrifuged at 7440 × g. The supernatant was discarded, and the precipitate suspended in 3 2 4 sterile water. A bacterial smear was prepared for microscopic examination. After confirming no 3 2 5 soil particles remained in the microbe solution, it was added to the drinking water of the MW mice. The water was changed using a fresh microbial mixture once a week. The soil sample and 3 2 7 microbes sample isolated from the soil underwent 16S rDNA high-throughput sequencing to 3 2 8 analyze their microbial compositions (Table S2). to minimize and alleviate the pain the animals may experience. Specifically, the health of the mice 3 3 5 was monitored every other day, and the weight measured weekly. The health status of mice was  Animal experiments were carried out in strict accordance with the guidelines of the Animal groups and PCoA was performed to show dissimilarities using QIIME. The Bray-Curtis 3 9 0 dissimilarity metrics between any two groups was calculated based on the metagenomic sequence 3 9 1 data and PCoA was performed using R version 3.2.3. Random forests analysis was performed as and analysis of variance (ANOVA). Raw sequence reads for all samples described above were deposited into the NCBI Sequence Read Funds for the Central Universities (grants no. 2242021k30014 and 2242021k30059). No competing interests were disclosed. References 4 1 0  N  o  o  r  a  ,  O  .  ,  L  a  s  s  e  ,  R  .  ,  A  l  i  n  a  ,  S  .  ,  H  a  n  n  a  ,  S  .  ,  P  i  i  a  ,  K  .  ,  J  e  n  n  i  ,  L  .  K  .  e  t  a  l  .  (  2  0  1  8  )  S  o  i  l  e  x  p  o  s  u  r  e  m  o  d  i  f  i  e  s   4  6  3   t  h  e  g  u  t  m  i  c  r  o  b  i  o  t  a  a  n  d  s  u  p  p  o  r  t  s  i  m  m  u  n  e  t  o  l  e  r  a  n  c  e  i  n  a  m  o  u  s  e  m  o  d  e  l  .   J  o  u  r  n  a  l  o  f  A  l  l  e  r  g  y  &   4  6  4   C  l  i  n  i  c  a  l  I  m  m  u  n  o  l  o  g  y   :  S  0  0  9  1  6  7  4  9  1  8  3  0  9  3  4  5  -.   4  6  5   R  i  e  d  l  e  r  ,  J  .  ,  B  r  a  u  n  -F  a  h  r  l  ä  n  d  e  r  ,  C  .  ,  E  d  e  r  ,  W  .  ,  S  c  h  r  e  u  e  r  ,  M  .  ,  W  a  s  e  r  ,  M  .  ,  M  a  i  s  c  h  ,  S  .  e  t  a  l  .  (  2  0  0  1  )   4  6  6   E  x  p  o  s  u  r  e  t  o  f  a  r  m  i  n  g  i  n  e  a  r  l  y  l  i  f  e  a  n  d  d  e  v  e  l  o  p  m  e  n  t  o  f  a  s  t  h  m  a  a  n  d  a  l  l  e  r  g  y  :  a  c  r  o  s  s  -s  e  c  t  i  o  n  a  l  s  u  r  v  e  y  .   4  6  7 T h e L a n c e t 3