Old Dog, New Trick: A Direct Role for Leptin in Regulating Microbiota Composition

The intestinal tract is the first barrier from the external to the internal environment. As such, it plays a key role not only in nutrient absorption but also in protection from toxins and immunity. However, the gut is not alone in this regulatory role. The mammalian intestinal tract is colonized by trillions of microorganisms collectively referred to as the microbiome. These bacteria have a symbiotic relationship with the host and contribute to many vital intestinal functions (1, 2). These functions include homeostasis of the immune system, including protection against pathogens, intestinal cell development and proliferation, xenobiotic metabolism, bone mineral density, behavior, and metabolism (1, 2).

That the gut microbiota plays a critical role in maintaining not only intestinal but also host homeostasis as a whole has been well described (3). Its role in disease has become more apparent over the last several years coinciding with advances in technology that allow for comprehensive high-throughput sequencing analysis of the microbiome. Data suggest that shifting of the microbiota populations alters the balance of the microbiome (dysbiosis), and this increases the risk of intestinal infection (4) and intestinal disorders such as irritable bowel syndrome and inflammatory bowel disease (5). Moreover, dysbiosis is implicated in diseases thought to be unrelated to intestinal function, including cardiovascular disease (6), obesity (2, 7), diabetes (8), autism (9), and even cancer (5). Conversely, correction of dysbiosis is also thought to be a mechanism for the drastic weight loss and metabolic improvements seen with bariatric surgery (10).

Given the wide-ranging implications for alterations in the microbiota, determining what regulates their differentiation is critical but, at present, is not completely understood. Variation of the microbiota is influenced in form or function by the introduction of probiotics (11), antibiotics (12), and diet (13) as well as development and aging (5), altogether demonstrating the role of the environment in regulating microbiome composition. Although genetics does play a role (14), internal pressures dictated by host physiology also influence the local colonization. Paneth cells, which are secretory epithelial cells, regulate development of the barrier between intestinal microbes and mucosal epithelial cells and also secrete antimicrobial peptides (15), gene-encoded antibiotics (16). Antimicrobial peptides, in turn, target intruding pathogens but also resident microbiota of the small intestine (17, 18), thus shaping the composition of the microbiota. Understanding regulation of Paneth cells and/or antimicrobial peptides could provide a new pipeline for therapeutic targeting of a wide range of chronic diseases.

A new study in this issue of Endocrinology by Rajala et al (19) indicates that leptin is a key player in regulating both antimicrobial peptides and microbiota composition. Most bacterial species of the microbiome, and the species that have received the most attention in regard to metabolic disease, belong to 2 phyla, Bacteroidetes and Firmicutes (7). A reduced ratio of Bacteroidetes to Firmicutes is associated with obesity in mice (7) and humans (20). However, because many external and internal pressures regulate microbiota composition, it is difficult to tell whether the changes in the microbiome came first or whether the disease influenced the microbiome. For example, obese humans and mice (2, 7) have increased Firmicutes relative to Bacteroidetes, but it is difficult to discern whether the change in the phyla distribution was the cause or consequence of obesity. Similarly, like dietary-induced obese animals, both leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice have increased Firmicutes relative to Bacteroidetes (7, 22). Although these animals are certainly obese, they also have defective leptin signaling. Thus, although these studies were important for establishing a role for obesity in regulating microbiota composition, they fail to advance understanding of the potential independent role of leptin in regulating microbiota composition.

The study by Rajala et al (19) found that mice with whole-body deficiency of the leptin receptor had a significant reduction in the mRNA expression of Paneth cell-specific antimicrobial peptides, specifically α-defensin 1, α-defensin 5, and defensin α related sequence 1-cr. To avoid the confounding effects of hyperphagia and obesity, the authors pair-fed the db/db mice with their wild-type siblings. Despite similar body weights to lean mice, pair-fed db/db mice still displayed a shift in fecal microbial composition toward a decrease in the Bacteroidetes to Firmicutes ratio. Thus, leptin receptor signaling has a direct role in regulating microbiota composition. Consistent with this, the pair-fed db/db mice also had significant changes in expression of various antimicrobial peptides, although the pattern of these changes was slightly different compared with the ad libitum-fed db/db mice. Specifically, the expression of α-defensin 1 and defensin α related sequence 1-r were significantly reduced in the lean pair-fed db/db animals, whereas there was no significant change in α-defensin 5. Similarly, the expression of regenerating islet derived-3 (reg3)β and reg3γ was similar between ad libitum-fed db/db mice and wild-type mice but was significantly reduced in pair-fed db/db mice. Thus, although leptin signaling is essential for production of some antimicrobial peptides, other antimicrobial peptides (reg3β and reg3γ) may be regulated by one of the many other factors that contribute to their secretion. Together these data demonstrate that leptin regulates antimicrobial peptide-encoding genes in the gut epithelium and that this in turn may explain the shift in the microbiome.

Previous work had demonstrated a role for intestinal epithelial cell leptin signaling in an intestinal infection of Entamoeba histolytica (22). To determine whether this receptor population was necessary for leptin-induced regulation of antimicrobial peptides, mice devoid of gut epithelial leptin receptors were next generated by crossing leptin receptor floxed animals to mice with cre recombinase driven by the Villin 1 promoter. Intestinal epithelial leptin receptor-knockout animals were similar to their littermate controls in body weight, food intake, fecal microbiota composition, and antimicrobial peptide expression. This leaves open the question as to which intestinal leptin receptor populations are responsible for leptin-induced regulation.

Because this was likely a perplexing finding, the authors then did a series of careful experiments to characterize the intestinal cell populations that contain the long form of the leptin receptor. Mice with cre recombinase driven by the long form of the leptin receptor were crossed with a cre-inducible tdTomato fluorescent protein reporter line, and immunohistochemistry was performed on intestinal sections. Based on this analysis, the leptin receptor was found on cells within the intestinal submucosa, not in the epithelium, and expression increased in density from the proximal gut to the colon; the latter finding was confirmed by quantitative PCR. The authors also demonstrated minimal expression of the leptin receptor in isolated gut epithelium using quantitative PCR and fluorescence-activated cell sorting. Moreover, the intestinal leptin receptor did not colocalize with several markers each for neurons, myeloids, myofibroblasts, or pericytes. However, they were found in the submucosa in close approximation to the vasculature, revealing a previously unknown population of leptin receptors. These data advance our understanding of leptin physiology on 2 fronts: 1) the careful characterization of a newly identified population of leptin receptors on perivascular cells within the submucosa and 2) the determination that intestinal leptin signaling is an essential component of host regulation of microbiota composition. Given that leptin signaling is crucial for regulating energy (23) and immune (24) homeostasis, it is interesting that it regulates the composition of the intestinal microbiome as well.

These data provide further support for the role of leptin as a unique bridge between nutrition and immunity. It is interesting to speculate that leptin's role as an adiposity signal to the brain (a signal that informs the central nervous system about the amount of stored fuel) is extended to the gut. Key questions remain, however. For example, does leptin signaling directly regulate antimicrobial peptides, which in turn regulates the microbiota, or does leptin signaling directly regulate the microbiota, which feeds back to regulate antimicrobial peptide secretion? Regardless, this kind of work, which brings together the fields of immunology and endocrinology, could be key for developing potential microbial-targeted therapeutics for the many chronic diseases associated with dysbiosis.