Gut microbiome communication with bone marrow regulates susceptibility to amebiasis

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens–colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens–colonized specific pathogen–free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.


Introduction
Commensal intestinal bacteria may protect from infection (1,2) by modulating bone-marrow production of innate immune effector cells including neutrophils and inflammatory macrophages (3)(4)(5). The host metabolome is influenced by the composition of the commensal gut microbiome, and is implicated in communicating and directing the development of innate immunity, to some extent, via bile acids (6). Primary bile acids produced by the host and secondary bile acids metabolized by the intestinal microbiota (e.g. deoxycholic and lithocholic acid), can act as signaling molecules, much like host damage-associated molecular pattern molecules (DAMPs) (6). Bile acids within the intestine may protect from intestinal pathogen infection (7).
Bile acid receptors are expressed in many cells implicated in innate immunity, are present in the myeloid lineage, and may impact expansion of these cells (8). Bone-marrow also has the ability to recognize bile acids (9,10). Epigenetic effects may result from signaling via bile acids, including inducing methyltransferase activity (11). This may explain in part how infection with one microorganism alters the inflammatory response to other pathogens, providing innate protection from infection with unrelated pathogens (12)(13)(14)(15).
Epigenetic changes, such as H3K27 and H3K4 methylation associated with promotor regions of innate inflammatory genes (16)(17)(18), have been implicated as a mechanism for this process. As such, commensal microbial metabolite alteration of H3K27 demethylase expression in innate immune populations might contribute to protection from infection (17,19). Host DAMPs that can be systemically induced by the microbiota have also been shown to be important in upregulating demethylase expression in myeloid cell lines and mouse bone-marrow (12,20). Collectively, these data suggest a role of serum soluble mediators, including secondary bile acids, induced by the microbiota in communicating to the bone-marrow to influence immunity to infection. We sought here to better understand the mechanism by which protective immunity induced by a metabolic product of the microbiota might occur during infection with a human intestinal pathogen.

Methods
Supplemental materials include full methods. Sequencing data is contained in the GEO repository under accession number GSE121503, the SRA under accession number PRJNA503904 and under SRA and linked via the dbGaP accession number phs001478.v1.p1.
Data is available in the manuscript and supplement. Design of the human cohort studies have been described (21,22) and all studies were approved by the Research and Ethical Review Committees of the icddr,b and the Institutional Review Boards of the University of Virginia. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Virginia. All experiments were performed according to provisions of the Animal Welfare Act of 1996 ( § 89.544).

Results and Discussion
Previous work suggested that murine commensals influence the inflammatory capacity of marrow derived cells (12,13). We hypothesized that components of the human gut microbiota might alter bone-marrow hematopoiesis to confer protection against unrelated pathogens such as Entamoeba histolytica (23,24). To explore this possibility, we first tested for human commensals associated with protection from amebiasis. Principal coordinates analysis of beta-diversity indicated that the microbiota of children with E. histolytica diarrhea differed significantly  (7,25,26). We hypothesized that these bacteria provide protection from Entamoeba. To test this hypothesis we introduced a member of the Lachnoclostridium genus, the human commensal bacteria Clostridium scindens (26) into the gut microbiome of susceptible CBA/J mice (27) and challenged them with the parasite E. histolytica.
C. scindens was significantly increased in the microbiota after gavage, and gut community structure was also altered ( Figure S5, A, B). Introduction of C. scindens to the gut microbiome provided protection from E. histolytica (Figure 1 D

Myeloid cell expansion may be influenced by cytokine production by CD8+T-cells (28) or
intestinal Tregulatory cells (29). Contribution of the acquired immune system to C. scindensmediated protection was tested by using RAG-1 -/mice, which lack B and T-cells. RAG-1 -/mice were also protected from E. histolytica when colonized with C. scindens (Figure 1

J, K)
indicating that protection did not require the acquired immune system.
The increase in gut neutrophils in response to Entamoeba infection in C. scindens colonized mice suggested that C. scindens may have altered innate bone-marrow populations that give rise to neutrophils. Therefore we examined hematopoietic progenitors in C. scindens colonized specific-pathogen-free mice (SPF) (Figure 2 A,  To explore this possibility, we examined transcriptional and epigenetic changes in marrow GMPs from C. scindens colonized mice. Gene enrichment analysis of RNA sequencing data suggested that genes associated with covalent modification of the histone H3 tail, such as the demethylase JMJD3, were enriched in mice exposed to C. scindens ( Figure S3  Adoptive transfer of bone-marrow from C. scindens colonized mice into mice not previously exposed to C. scindens was sufficient to provide protection from E. histolytica (Figure 3 A) as well as recapitulate the increase in marrow GMPs (Figure 3 B) and intestinal neutrophils (Figure 3 C). In contrast, previous epithelial exposure to C. scindens was not sufficient to provide protection from ameba in irradiated mice ( Figure 3A).We also noted an overall increase in GMPs in the mice post bone-marrow transplant: however, this increase was controlled across all groups and was likely a response to irradiation (32). We concluded that alterations in radiosensitive marrow hematopoietic cells caused by gut exposure to C. scindens were sufficient to confer protection to a later E. histolytica challenge.
We next explored how intestinal colonization with C. scindens could be altering GMPs in the bone-marrow. C. scindens is known to be capable of 7α-dehydroxylation of bile acids in the intestine (26). Gavage and colonization with another human mucosal anaerobic bacterium lacking 7α-dehydroxylation activity did not induce protection from Entamoeba ( Figure S5, C-E). Colonization of mice with C. scindens was sufficient to increase serum levels of the secondary bile acid deoxycholic acid (DCA, a product of 7α-dehydroxylation of cholic acid) in SPF and in gnotobiotic mice (Figure 4 A, Figure S2 A, B ). Absolute levels of DCA were lower in gnotobiotic mice in both groups than in SPF however. This is perhaps due to the lack of other members of the microbiota producing products upstream of 7α-dehydroxylation (33). DCA was also increased in children (from two independent cohorts) protected from E. histolytica (Figure 4 B, Figure S1 A, B). We concluded that DCA in plasma was positively correlated with protection  (Figure S4 A) as well as induction of intestinal neutrophils and protection from E. histolytica (Figure S4 B, C). This suggests H3K27 demethylase activity may contribute to gut to marrow communication by C. scindens. JMJD3 is an H3K27me3 demethylase (17); however, we also observed changes in H3K4me3 in the promoter region of CEBPB. JMJD3 has recently been shown to impact H3K4me3 levels in