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dcyphr | Enteric Nervous System0Derived IL-18 Orchestrates Mucosal Barrier Immunity

Abstract

Mucosal barrier immunity in the intestine is essential for maintaining the commensal microflora (friendly gut bacteria) and fighting bacterial infection. Previously, immune and epithelial cells were thought to maintain this balance between harboring the commensal microflora and fighting infection. In this study, the researchers show that the enteric nervous system (ENS), or neurons in the gastrointestinal tract, plays an important role in this balance. The researchers found that the ENS governs the antimicrobial protein (AMP) response, which helps fight against infection. The researchers used confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) to observe that intestinal neurons produce IL-18, a cytokine. Deletion of IL-18 production from the ENS, but not from immune or epithelial cells, caused mice to be susceptible to Salmonella typhurium (S.t.) infection, indicating that IL-18 production by the ENS is essential and non-redundant. In addition, RNA sequencing and single-cell sequencing showed that IL-18 produced by the ENS is required for AMP production. Together, these results show that IL-18 produced by the ENS signals to and controls intestinal immunity, and has important implications for the mucosal barrier and fighting off infection.

Aims

The researchers wanted to understand how the enteric nervous system influenced immune responses in the intestine.

Introduction

The intestinal mucosal barrier protects against bacterial infection while maintaining a symbiotic relationship with commensal microflora. It is well known how immune and epithelial cells maintain this balance, but little is known about the influence of the enteric nervous system (ENS).

Patients with Hirschsprung disease have an absence of nerves in the bowel. These patients are at risk of developing serious and life-threatening infection of the bowel. This infection correlates with dysregulation of intestinal mucus and imbalances in the commensal microflora, suggesting a connection between the ENS and intestinal mucosal barrier. Previous studies have concluded that ENS cells can respond to patterns associated with infection. This suggests that the ENS may play a key role in regulating mucosal barrier immunity.

Interleukin 18 (IL-18) has emerged as an important cytokine in the immune response. It encourages inflammation and is required to combat invasive bacterial infections, such as Salmonella. IL-18 deficient mice have shown imbalances in the commensal microflora and decreases in antimicrobial protein (AMP) production. Understanding of this cytokine outside of immune and epithelial cells is largely unknown. In this study, the researchers found that immune and epithelial cells were redundant sources of IL-18, which drives AMP production and protection against Salmonella infection. The results demonstrate that the ENS plays a critical part in the immune response, and is essential for coordinating the balance between harboring the commensal microflora and combating infection.

Results

Epithelial Cell- and Immune Cell-Derived IL-18 Does Not Protect against Enteric S.t. Infection

Epithelial cells and myeloid cells (which give rise to many kinds of immune cells) are thought to be the major sources of IL-18 production. To test this, the researchers crossed wild-type mice with mice that have IL-18 deletion in myeloid and epithelial cell lineages. Wild-type and mutant mice were subjected to Salmonella infection. Absence of IL-18 in both epithelial and myeloid cells did not affect susceptibility to infection, and mice were healthy after infection. These results suggested that epithelial- or immune-derived IL-18 are required for protection against infection.


Enteric Neurons Express IL-18

To investigate whether the ENS produces IL-18, the researchers used confocal microscopy for IL-18 reactivity. The researchers found a subset of neurons in the ENS that produced IL-18, indicating that enteric neurons are novel producers of IL-18 in the gastrointestinal tract.


Enteric Neuronal IL-18 is Protective against S.t. infection

To investigate the role of ENS-derived IL-18 on intestinal immunity, the researchers bred wild-type mice and mice with genes that allowed deletion of IL-18 from ENS cells. Loss of IL-18 did not affect the commensal microflora in progeny mice. The researchers then infected progeny mice with Salmonella and observed that mice without ENS-derived IL-18 became infected and sick, and showed increased prevalence of bacteria in the gastrointestinal tract. On the other hand, when wild-type mice were infected, the mice were more healthy than mice without ENS-derived IL-18. These results showed that ENS-derived IL-18 protects mice from bacterial infection.


Enteric neuronal IL-18 is a Specific Driver of Goblet Cell Antimicrobial Protein Expression

Since it was not obvious how ENS-derived IL-18 protects against Salmonella, the researchers sought to understand what kind of signaling events that occur in mice deficient in ENS-derived IL-18 compared with mice deficient in epithelial and immune cell-derived IL-18. The researchers conducted RNA-seq on colon tissue of both wild-type and mutant mice. Analysis showed that distinct genetic expression was specifically regulated by different sources of IL-19 (neuronal, epithelial, and myeloid). The researchers compared these genes and found that bactericidal and antimicrobial genes were exclusively reduced in mice lacking ENS-derived IL-19. This suggested that the source of IL-18 has significant effects on what role it has in the intestinal immune response.

Analysis also showed that ENS-derived IL-18 was specifically promoting AMP production in the colon. The researchers found that these AMPs were produced by goblet cells, which secrete mucus in the intestine. These results showed that ENS-derived IL-18 drives intestinal AMP production through indirect signaling mechanisms.


Neuronal IL-18-Driven AMP Expression Prevents Bacterial Infiltration and Infection

The researchers also wanted to understand whether deficiency of ENS-derived IL-18 led to targeted loss of AMPs or if AMP loss was caused by other disturbances in the intestine. Using histological staining and immunofluorescence studies, the researchers found no major defects in the structure or immunological environment in the intestine.

The researchers also investigated the impact of ENS-derived IL-18 on the commensal microflora.  They observed no significant alterations. However, they did observe that in mutant mice, there was increased bacterial infiltration from the commensal microflora into the intestine. These results revealed that ENS-derived IL-18 signals AMP production that is necessary to prevent bacteria from the commensal microflora infiltrating the intestine.

The researchers tested whether decreases in AMP production explained why mutant mice were more susceptible to Salmonella infection. They found that bacterial killing was significantly reduced in mutant mice compared to wild-type mice. These results demonstrate that reduction in AMPs in mice deficient in ENS-derived IL-18 causes an inability to ward off infection.

Discussion

In this study, the researchers found that IL-18 produced by the ENS is necessary for protection against bacterial infection. Expression of IL-18 in the ENS non-redundantly signals for AMP production in goblet cells, which helps prevent the commensal microflora from invading the intestine and, during infection, kills bacteria. The researchers also found that epithelial and immune cell-derived IL-18 is completely unnecessary for AMP expression. The results of the study demonstrated that the source and location of IL-18 also influence its effects. 

Science’s understanding of how the nervous system influences intestinal immunity has grown rapidly in recent years. The findings of this study show that neuron-produced IL-18 is an important driver of the immune response. Based on these results, the researchers suggest that targeting neurons to enhance AMP production may be used to limit infection in patients suffering from deficiencies in barrier immunity.