Creeping fat.

https://doi.org/10.58974/bjss/azbc018

Creeping fat (CrF), migrating mesenteric adipose tissue (MAT), is a hallmark of Crohn’s Disease¹ (CD) frequently associated with intestinal fibrosis. Surgeons operating for CD recognize CrF not only as a disease-specific phenomenon, but also as a technical challenge. Mesenteric transection is complicated in CrF as vessels retract with thickened, fibrotic fatty tissue resulting in haemorrhage and making haemostasis more difficult. Despite clinical familiarity with CrF however, its aetiology remains largely unknown. Additionally, it is unclear whether CrF represents an adaptive response in an attempt by the mesentery to seal off a “leaky” inflamed gut or is a pathological one, worsening the cycle of CD-related intestinal inflammation, scarring, stenosis, and ultimately development of surgical complications such as obstruction, perforation and fistula.  

This study sheds new light on CrF, explaining how this adipose tissue transitions from the traditional storage role to an active component, generating an adipogenic, fibrotic, and inflammatory environment. The authors hypothesize that CrF is a both a response to translocated gut microbiota and a driving force in fibrosis. They investigate these theories by identifying the bacterium Clostridium innocuum as a signature organism in CrF, characterizing CrF as a milieu distinguished by immune response and fibrosis, determining that C. innocuum gavage promotes this milieu and recapitulates CrF in gnotobiotic mice, and demonstrating in vitro that selective promotion of M2a macrophages may be the means by which C. innocuum drives fibrosis.

The authors identify and focus on C. innocuum by first performing a metagenomic sequencing analysis on surgical samples of ileal tissue in CrF, grossly normal CD MAT, ulcerative colitis (UC) MAT, and healthy controls. The authors found that healthy MAT contains bacteria, suggesting that bacterial translocation to MAT is not in itself pathogenic. CD MAT, however, contained higher numbers of bacteria, but with lower biodiversity than healthy controls, consistent with mucosal data and previous studies^2,3. 16S-RNA sequencing comparison to healthy MAT and UC-MAT identified a CD-specific expansion of the Erysipelotrichae lineage including C. innocuum. To assure the sequencing findings represented viable bacteria, the authors cultured MAT-derived bacterial isolates. Five bacteria were isolated as CD-specific and viable after cultivation.Among those, C. innocuum was most frequently isolated and selected for further study.  A whole genome sequencing and comparative genomics study of C. innocuum revealed a conserved core of genes advantageous for translocation in adipose-like environments, such as protection against oxidative damage, cell motility, lipid detoxification, and evasion from immune functions and strain divergence between C. innocuum isolated from MAT and that isolated from mucosa. Factors potentially promoting viability in creeping fat also include type IV pili and mobility, lipid catabolism, and preference for b-hydroxybutyrate, a product of fatty acid oxidation.

The authors investigated permissiveness of MAT to bacterial translocation by comparing intestinal permeability across healthy controls, CD, and UC. Expression of most tight junction markers was lower in CD. However, plasma circulating levels of bacterial products were significantly lower in CD in comparison to UC, but largely comparable to healthy controls, perhaps indicating that creeping fat functions to contain bacteria in the context of impaired innate intestinal barrier function.

The authors then characterized the CrF milieu in terms of gene expression and cellular components in comparison to healthy MAT, normal CD-MAT, and UC-MAT. Bulk RNA sequencing unsurprisingly identified upregulation of adipogenesis-promoting genes in CrF, but the most overexpressed genes were those that promote phagocytosis, extracellular matrix (ECM) organization, B- and T-cell differentiation, and cellular response to bacterial products. This underpins the idea that CrF’s predominant function is immune response and ECM development. Single cell RNA-seq revealed that CrF is well-populated with immune cells and pro-fibrotic progenitor cells compared to healthy MAT. Non-CrF CD-MAT in general was intermediate between healthy MAT and CrF.

Finally, the authors tested the ability of C. innocuum to recapitulate the observed features of human CrF in a model organism and in vitro. First, a gnotobiotic mouse model served to demonstrate C. innocuum in action; the bacterium successfully translocated from the gut to MAT with and without the presence of inflammation induced by dextran sodium sulfate (DSS) colitis. The mice gavaged with C. innocuum developed a dramatic increase in mesenteric adiposity and demonstrated an upregulation of genes of adipogenesis and ECM collagen production. Similar to human findings, C. innocuum gavaged mice had lower circulating levels of plasma bacterial products than control gnotobiotic mice, but these plasma levels were similar between groups following DSS treatment.  In vitro, the authors studied macrophages as a potential immune cell mediator between C. innocuum and the adipogenic/fibrogenic CrF phenotype. C. innocuum lysates caused the macrophages of healthy volunteers to differentiate into a pro-fibrotic M2a subtype. This response was pronounced compared to treatment with lipo-polysaccharide (LPS), mixed CD bacteria, and IL-4. Finally, the authors exposed fibroblasts and adipocyte stem cells derived from CD-MAT to C. innocuum lysate or conditioned media from C. innocuum-exposed macrophages. The media, but not the lysate, significantly increased production of ECM-collagens. 

In the case of creeping fat seen in CD, the gut-derived bacterium C. innocuum translocates to MAT in human tissue and recapitulates CrF in a mouse model. Innate features of the bacterium likely support its survival in MAT and strain divergence suggests selective pressure in CrF. In vitro data suggest macrophages may act as immune cell mediators between C. innocuum and fibrosis.Bulk RNA-Sequencing and scRNA-seq reveal CrF as a highly immunogenic, adipogenic, and pro-fibrotic environment.

This study sheds new light on CrF, a clinical feature of CD long-recognized but poorly understood. For surgeons who have long struggled with this tissue without an explanation for its existence, this study provides some initial satisfaction of intellectual curiosity and clinical frustration. Many questions remain, however, particularly whether CrF is an adaptive or deleterious response. The fibrotic response of adipose may confer a benefit, potentially containing enteric pathogens and toxins. However, in the context of persistent inflammation, an uncontrolled expansion of fibrosis may begin to harm underlying bowel wall, ultimately leading to stenosis and obstruction. Thus, the authors suggest that prophylactic treatment against an unmitigated fibrosis cascade could be developed by targeting C. innocuum and its fibrotic response to intestinal damage in high-risk patients with CD.