Limosilactobacillus reuteri promotes the expression and secretion of enteroendocrine- and enterocyte-derived hormones

Abstract

Observations that intestinal microbes can beneficially impact host physiology have prompted investigations into the therapeutic usage of such microbes in a range of diseases. For example, the human intestinal microbe Limosilactobacillus reuteri strains ATCC PTA 6475 and DSM 17938 are being considered for use for intestinal ailments including colic, infection, and inflammation as well as non-intestinal ailments including osteoporosis, wound healing, and autism spectrum disorder. While many of their beneficial properties are attributed to suppressing inflammatory responses in the gut, we postulated that L. reuteri may also regulate hormones of the gastrointestinal tract to affect physiology within and outside of the gut. To determine if L. reuteri secreted factors impact the secretion of enteric hormones, we treated an engineered jejunal organoid line, NGN3-HIO, which can be induced to be enriched in enteroendocrine cells, with L. reuteri 6475 or 17938 conditioned medium and performed transcriptomics. Our data suggest that these L. reuteri strains affect the transcription of many gut hormones, including vasopressin and luteinizing hormone subunit beta, which have not been previously recognized as being produced in the gut epithelium. Moreover, we find that these hormones appear to be produced in enterocytes, in contrast to canonical gut hormones which are produced in enteroendocrine cells. Finally, we show that L. reuteri conditioned media promotes the secretion of several enteric hormones including serotonin, GIP, PYY, vasopressin, and luteinizing hormone subunit beta. These results support L. reuteri affecting host physiology through intestinal hormone secretion, thereby expanding our understanding of the mechanistic actions of this microbe.

Keywords: GIP; Lactobacillus; PYY; adipolin; enterocyte; enteroendocrine; hormone; kisspeptin; luteinizing hormone; small intestine; vasopressin.

Figure 1.

Induced and uninduced NGN3-HIOs differentially respond to L. reuteri treatment. A) Overview of RNA-Seq experiment. First, L. reuteri conditioned media was prepared by growing L. reuteri 6475 and 17938 in LDM4 to mid-log phase. The bacterial cells were spun out, the resulting conditioned media brought to neutral pH, and then filtered through a 0.22 μm filter. The conditioned media were then lyophilized and resuspended in HIO differentiation media. These treatments were then placed into uninduced or induced NGN3-HIOs in transwells for three hours. Third, the organoid cells were harvested, and isolated RNA was sent for RNA-Seq. Created with BioRender.com. B) Principal coordinate analysis of transcriptomic data from NGN3-HIOs induced or not induced and treated with L. reuteri 6475, 17938, or LDM4 media control. Ellipses for illustration purposes are modeled from the data following a t-distribution. C) Enriched functional categories of differentially expressed genes in L. reuteri treatments over media alone. U6475 is L. reuteri 6475 vs media control in uninduced NGN3-HIOs. I6475 is L. reuteri 6475 vs media control in induced NGN3-HIOs. I17938 is L. reuteri 17938 vs media control in induced NGN3-HIOs. Some functional groups are listed as belonging to two categories (see Supplemental Table 3 for further details).

Figure 2:

Hormone genes differentially expressed by L. reuteri. DEGs annotated as having hormonal function are shown. The genes are annotated with their function, whether they are secreted, a receptor, or intercellular, and what cluster they belong to as in Supplemental Figure S4. The graph shows the log₂ fold change expression of the gene for the indicated comparison. The bars are colored using the log₁₀ scaled mean GeTMM counts to illustrate how abundantly expressed the gene is. Transparent overlays are used on genes not differentially expressed for the given comparison. Comparisons shown: U6475-ULDM4, L. reuteri 6475 on uninduced HIOs compared to LDM4 media control; I6475-ILDM4, L. reuteri 6475 on induced HIOs compared to LDM4 media control; I17938-ILDM4, L. reuteri 17938 on induced HIOs compared to LDM4 media control; I6475-I17938 L. reuteri 6475 compared to L. reuteri 17938 on induced HIOs; ILDM4-ULDM4, LDM4 media control on induced versus uninduced HIOs; I6475-U6475, L. reuteri 6475 on induced versus uninduced HIOs. For each, positive fold changes indicate genes upregulated by the condition listed first.

Figure 3.

L. reuteri promotes the secretion of known enteroendocrine-derived intestinal hormones. A) 1) In order to measure the release of intestinal hormones from human intestinal organoids (HIO), L. reuteri conditioned media is generated from mid-log phase cultures of L. reuteri. These cultures are pH neutralized and rendered cell-free. 2) L. reuteri conditioned media is then placed onto NGN3-HIOs plated on transwells that are differentiated but not induced for NGN3 or induced for NGN3. 3) Following an incubation on the HIOs, the supernatant is collected and secreted hormones are measured by ELISA or Luminex assay. Created with BioRender.com. Secreted amylin (B), C-peptide (C), ghrelin (D), GIP (E), PP (F), and PYY (G) measured from uninduced and induced NGN3-HIOs in response to L. reuteri 6475 or 17983 conditioned media. Hormones in B-G were measured on the apical side only of the transwell. In B-G, batches A and B from the RNASeq experiment were pooled so each point on the plot is the result from two organoid batches pooled together. H) Serotonin released from the apical or basolateral side (as indicated) from uninduced and induced NGN3-HIOs in response to L. reuteri 6475 or 17983 conditioned media. In H, shape denotes independent batches of organoids. Only p-values <0.1 are shown with p<0.05 being considered significant. Significance was determined with a Dunnett’s Test.

Figure 4:

L. reuteri promotes the secretion of enterocytic hormones. A) Gut Cell Atlas annotated UMAP of the adult jejunum (adapted from Danhof et al 2023), highlighting the enteroendocrine marker CHGA (B), the enterocyte marker SI (C), vasopressin (AVP, D), luteinizing hormone subunit beta (LHB, E), and adipolin (C1QTNF12, F). G) Adipolin visualized in human jejunal tissue. Scale bar represents 50 μm. Secretion of H) vasopressin and I) luteinizing hormone subunit beta and J) the lack of secretion of adipolin from whole human jejunal tissue using the method shown in Figure 3A except with ex vivo human jejunal intestinal tissue. Shape represents unique human intestinal donors. Significance was determined using a linear mixed model with p <0.05 considered as significant.