Stable serum peptidoglycan fragment levels do not support leaky gut in the acute phase or at one month following “mild” traumatic brain injury: A preliminary study

TL;DR

This study found no evidence of increased gut leakage after mild traumatic brain injury, but observed later immune system activation unrelated to gut bacteria.

Contribution

First preliminary investigation linking NOD2-mediated inflammation to mTBI without evidence of leaky gut.

Findings

Serum MDP levels remained stable in mTBI patients and controls at acute and one-month timepoints.

NF-κB signaling in NOD2-expressing cells increased at one month post-injury, unrelated to MDP levels.

Increased NF-κB activity correlated with higher IL-6 and IL-10 levels in the acute phase.

Abstract

The gut-brain axis is increasingly recognized as contributor to the pathophysiology of brain disorders, in part through its influence on inflammation. Impaired gut health can lead to so-called leaky gut, allowing bacterial cell wall fragments such as peptidoglycan-derived muramyl dipeptide (MDP) to translocate to the circulation. MDP can activate microglia, key mediators of neuroinflammation, via the intracellular receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Although neuroinflammation is a hallmark of “mild” traumatic brain injury (mTBI), the link between leaky gut and mTBI remains largely unexplored.

This preliminary prospective study investigated whether mTBI leads to increased MDP levels and NOD2 activation in the acute phase (N = 246; median 106 min post-injury) and at a ∼1-month follow-up visit (N = 140; median 32 days) in an emergency department cohort, relative to healthy controls (HC; N = 31, with N = 26 at ∼1-month). Serum MDP concentration was measured using an indirect competitive ELISA. Engagement of the pro-inflammatory nuclear factor (NF)-κB pathway was measured in NOD2-transfected cells. Additionally, blood interleukin (IL)-6 and IL-10 levels were quantified. Clinical outcome was measured at six months post-injury using the extended Glasgow Outcome Scale and a symptom questionnaire.

Linear mixed effects models showed that concentrations of MDP remained stable across visits in both mTBI and HC (P = 0.62), with no significant main effect of group (P = 0.16) or group × visit interaction (P = 0.25). In contrast, for engagement of NF-κB signaling in NOD2-expressing cells, a significant group × visit interaction (P = 0.004) was observed, with an elevation in mTBI relative to HC at ∼1-month post-injury (P = 0.01, Cohen's d = 0.48), but not in the acute phase (P = 0.22, d = 0.22). This elevation was associated with higher IL-6 (β = 0.16, P = 0.02) and IL-10 (β = 0.17, P = 0.006) levels in the acute phase. No associations with clinical outcome were observed.

In conclusion, our preliminary null findings for serum MDP do not directly support the emergence of leaky gut in either the acute phase or at ∼1-month following mTBI. However, transient increases in MDP occurring during the first month cannot be ruled out based on our findings. Increased engagement of the NF-κB pathway in NOD2-expressing cells likely reflects (damage-associated) mechanisms other than MDP. Assessment of NF-κB signaling may serve as a useful marker for studying chronic (neuro)inflammation following mTBI, complementing interleukin responses in the acute phase, an avenue warranting further investigation.

•Serum peptidoglycan fragment levels did not change <24 h or ∼1-month post mild TBI.•The innate immune receptor NOD2 in microglia binds peptidoglycan fragments.•We quantified NF-κB activity within human embryonic kidney cells expressing NOD2.•∼1-month post-injury serum increased NF-κB activity in these cells.•This likely reflects peptidoglycan-independent neuroinflammatory mechanisms.

Serum peptidoglycan fragment levels did not change <24 h or ∼1-month post mild TBI.

The innate immune receptor NOD2 in microglia binds peptidoglycan fragments.

We quantified NF-κB activity within human embryonic kidney cells expressing NOD2.

∼1-month post-injury serum increased NF-κB activity in these cells.

This likely reflects peptidoglycan-independent neuroinflammatory mechanisms.

Figures