Examining the immunomodulatory effects of the novel probiotic Bacillus subtilis DE111 2

: Probiotics make up a large and growing segment of the commercial market of dietary 18 supplements and are touted as offering a variety of human health benefits. Some of the purported positive impacts of probiotics include, but are not limited to, stabilization of the gut microbiota, prevention of gastrointestinal disorders and modulation of the host immune system. Current research suggests that the immunomodulatory effects of probiotics are strain specific and vary in mode of action. Here, we examined the immunomodulatory properties of Bacillus subtilis strain 23 DE111 in a healthy human population. In a randomized, double blind, placebo-controlled four-week intervention, we examined peripheral blood mononuclear cells (PBMCs) at basal levels pre- and 25 post-treatment as well as in response to stimulation with bacterial lipopolysaccharide (LPS). We 26 observed an anti-inflammatory effect of B. subtilis , manifested as a decrease in immune cell 27 populations within the basal state along with an increase in anti-inflammatory immune cells in 28 response to LPS stimulation. Overall gastrointestinal health, microbiota, and circulating and fecal 29 markers of inflammation and gut barrier function were largely unaffected by DE111 treatment. 30 These data suggest that the novel probiotic B. subtilis DE111 may have clinical applications in 31 modulating immune homeostasis via anti-inflammatory mechanisms. as determined via alteration 69 in specific immune cell populations. We tested this hypothesis using a parallel arm, double-blind, 70 randomized, placebo-controlled intervention study. Our primary outcomes included flow cytometric 71 analysis of immune cell populations from cultured peripheral blood mononuclear cells (PBMCs), 72 both with and without stimulation by bacterial lipopolysaccharide (LPS). Secondary outcome 73 measures included examining the effects of B. subtilis on perceived gastrointestinal health, gut 74 microbiota profiles, and circulating and fecal markers of inflammation and immune function. increases CD25+ and CD25+FoxP3+ well CD4+CD8+ T cells after LPS T cell subsets act to resolve modulate


36
The human GI tract houses roughly 3.8 X 10 13 microorganisms comprising up to 1,000 different 37 species (1). These microorganisms function to aid in digestion, protect the host from pathogens, and IgA production, competitive exclusion of pathogenic bacteria, and generation of bioactive

153
Using 16s rRNA sequencing, we next sought to determine whether DE111 consumption altered 154 the community structure of the gut microbiome. We did not detect any significant differences in 155 observed or estimated microbial richness (Supplemental Table 1) or in Bray-Curtis distances between 156 treatment groups or within a given treatment over time ( Figure 3C).

198
In order to better understand how DE111 treatment impacted immune cell responses, we were not significantly altered with LPS-stimulation (ie. response ratio of ~1; data not shown).

202
However, CD25+ and CD25+FoxP3+ T regulatory cell counts were significantly increased in the 203 DE111 treatment group, both from baseline levels and at the final timepoint between the two 204 treatment groups ( Figure 6A,B; Table 3). Similarly, DE111 treatment led to a significant increase in 205 CD4+CD8+ double positive T cells both within and between treatment groups ( Figure 6C; Table 3).

206
In contrast, CD8+ cytotoxic T cells were reduced after DE111 treatment compared to the placebo 207 group ( Figure 6D, Table 3). There were no significant differences in the percent proliferating cells C. D.

253
It is also important to note that most immune cells found in peripheral circulation are naïve 254 lymphocytes and myeloid cells. When myeloid cells activate naïve lymphocytes via antigen 255 presentation and cytokine stimulation, the lymphocytes migrate to the lymph nodes as effector cells.

256
Our observed decreases in these T cell subsets, therefore, could reflect an anti-inflammatory effect of 257 DE111 via decreased cell activation resulting in decreased overall circulating T cells. However, 258 without measurement of specific myeloid and T cell activation markers respectively, these 259 hypotheses are based on speculation.

260
In addition to assaying basal cell counts before and after probiotic treatment, we also sought to 261 determine how DE111 impacted the immune response to a pro-inflammatory challenge. To test this,

313
The results from our secondary outcome measures, specifically safety and tolerability of B.

314
subtilis DE111 intake, as well as its overall impact on microbiota stability, were largely confirmatory.

315
For example, a study that administered five million CFU/day of B. subtilis DE111 to 41 healthy adult 316 participants reported after ~20 days of consumption that blood metabolic parameters remained 317 within normal ranges and there were no significant gastrointestinal disturbances or alterations to 318 bowel movements (19). In addition, their reports of increased fecal counts of B. subtilis is consistent 319 with the treatment-associated increases that we report in our qPCR data and support that these treatment changes in taxa richness, although diversity did increase with treatment. While we did not 326 see concurrent changes in bacterial diversity, this may be due to the differences in age of the study tolerable, and while it may alter specific taxa, it does not disrupt the general homeostasis of the gut 330 microbiota.

331
A limitation of the current study was the use of cryopreserved PBMCs for assessing immune cell  age with a BMI of 20 to 34.9 kg/m2 were recruited into the study (Figure 1). Participants were 357 recruited from the Fort Collins, CO area by email, social media platforms, fliers, and word of mouth.

358
Participants were prescreened for eligibility through a phone interview. Those who met the inclusion 359 criteria were scheduled for an onsite screening visit to confirm eligibility. Exclusion criteria included 360 the following: A) those under 18 or over 65 years of age; B) BMI outside the range of 20-34.9 kg/m2); 361 C) pregnancy or breastfeeding; C) current prescription medication use; and D) use of antibiotics in 362 the last 2 months. Qualified participants were asked to maintain their regular exercise and dietary 363 habits throughout the study, abstain from supplemental pre-or probiotics, and to limit alcohol 364 consumption to one drink per day for women and two drinks per day for men, or no more than seven 365 drinks per week. Participants were randomized and enrolled into different treatment groups, fortyseven of which were enrolled into a separate study under the same protocol (Protocol #19-9145H). Of the total enrolled participants (n=94), 44 completed the currently outlined study.

368
Participants were asked to fast for eight hours (including no caffeine, soda, tea, etc.) and abstain 369 from exercise for 12 hours prior to arrival in the clinic. Additionally, participants were asked to delay 370 consumption of any medication or dietary supplements for 24 hours prior to the study visit.

371
Participants arrived at the FNCRL for their baseline visit to undergo sample collections (blood and 372 stool) and analysis procedures (weight/height, blood pressure, pulse wave and endothelial function