Final Report:  Oral Microbiome and Inflammation in Pediatric Headache

Summary

Headache disorders such as migraine and post-traumatic headache cause significant disability in the pediatric population. Our limited understanding of the factors that affect headache severity results in unmet treatment needs. Dysbiotic oral microbiome and inflammation in the oral cavity are plausible mechanisms of headache exacerbation. The oral cavity is richly innervated by branches of the trigeminal nerves, the nerves that mediate headache. Lipopolysaccharide (LPS) from oral bacteria is a source of inflammation that can sensitize the trigeminal nerves. Our hypothesis was that microbiome dysbiosis (microbiota that differ from the composition of the microbiota in healthy individuals) and elevated levels of LPS in saliva are associated with more severe headache and vary in patients with different types of headache. We tested this hypothesis by analyzing oral microbiota and LPS in saliva from children and adolescents with headache and healthy controls.

In Aim 1, we used 16S rRNA gene sequencing to characterize the oral microbiome profile associated with headache severity and specific headache groups. In Aim 2, we measured LPS levels in saliva samples and tried to identify if any headache characteristics are associated with higher levels of LPS in saliva.

We found differences in the oral microbiome composition depending on the severity of the headache. We identified higher levels of the genera Rothia in the sputum of patients with more severe headache. These nitrate-reducing bacteria have been previously reported elevated in migraine patients. Elevated levels of Rothia could increase nitric oxide production, which could in turn exacerbate headaches. The correlation to headache severity we found strengthens the argument for a role of oral microbiome in migraine physiology. We found no difference in LPS levels between groups or in relation to headache severity. Our limited testing yielded no evidence of increased oral inflammation in headache patients.

Hypothesis v. Findings

Aim 1. To characterize the oral microbiota profile in different headache disorders based on headache severity and phenotypes. Microbiome dysbiosis has been implicated in multiple conditions, but its role as a potential exacerbating factor in headache disorders has not been investigated. We hypothesized that dysbiosis and relative abundance of specific bacterial community structures will be associated with more severe headaches and specific phenotypes.

We performed 16S rRNA gene sequencing in oral samples from controls, migraine patients, post traumatic headache patients and controls. Both the amount and composition of bacteria and relative abundances of bacteria were different in the oral wash vs sputum samples. When we analyzed the different species of bacteria in both sputum and oral wash samples in regard to the severity of the headaches, we found that Actinobacteria Rothia levels were increased while Fusobacterua Leptotrichia were decreased in the higher severity groups.

The analysis of beta-diversity in our samples confirms that both sample types are different enough that the analysis need to be separate and results cannot be combined. On the other hand, the fact the differences observed in alpha-diversity trends and Rothia and Leptotrichia abundance changes replicate in both sample types makes these results more robust.

Aim 2. To identify the specific headache characteristics that are associated with higher levels of LPS in the oral cavity. LPS induces synthesis and release of proinflammatory cytokines. More importantly, LPS from oral bacteria sensitizes trigeminal neurons in vitro. We had hypothesized that severe and frequent headaches are associated with elevated levels of LPS and proinflammatory cytokines in the oral cavity.

LPS levels were very low in our saliva sample and we found no difference in the LPS concentrations between the different groups or in relation to headache severity. We have banked saliva samples to test cytokine levels in these patients in the future.

What this Research Means to You

Our hypothesis proposed that microbiome dysbiosis and elevated levels of LPS in saliva would be associated with more severe headache and different types of headache. This hypothesis seems to hold true where it comes to the changes in oral microbiome. The changes in Rothia levels are consistent with previously reported high levels of this bacteria in migraine patients.  This newly reported correlation to headache severity strengthens the argument for a role of oral microbiome in migraine physiology.

We had hypothesized that this role would be through LPS induced inflammation. We have not seen evidence of increased oral inflammation in headache patients. Even though our testing was limited, it seems more plausible that the oral microbiome may be playing a role in headache through the reduction of nitrate into nitrite, increasing the potential of NO production.

A sample of saliva is easily collected in the clinic and the characterization of the patient’s oral microbiome could open the possibility of complementary interventions that could improve the patient’s quality of life. These preliminary data also open the door to incorporate the analysis of oral microbiome in other research.