Oral bacteria can enter into the systemic circulation through inflamed gingiva and thereby affect peripheral organs and the CNS. In mice, oral administration of Campylobacter jejuni activated visceral sensory nuclei in the brainstem that processed gastro-intestinal sensory information; Porphyromonas gingivalis, an oral pathogen contributing to the development of chronic periodontitis, may be a risk factor for developing amyloid-beta plaques, cognitive impairment, and dementia. Further, exposure to viral or bacterial pathogens upregulates neuronal Ab expression in nontransformed cell culture models and wild-type rat brains, which may represent a naive antimicrobial defense response. Cannabis smoking alters the oral environment and produces numerous chemicals that directly interact with oral bacteria. Some of the chemicals are toxicants and may perturb the oral microbial ecology.In this study, we found that saliva Actinomyces, Veillonella, Megasphaera, and Streptococcus bacteria were increased, and Neisseria bacteria were decreased in cannabis smokers compared to non-smokers. Two Actinomyces species bacteria and one control Neisseria species bacterium were inoculated to the B6 mice via oral inoculation. A. meyeri administration resulted in reduced global mouse activity, macrophage infiltration,trim tray pollen and increased Ab 42 protein production in the brain.
Non-smoking controls were recruited from the Medical University of South Carolina and University of Connecticut by advertisement on the campus and chosen by self-report of non-drug use. Cannabis-smoking individuals with cannabis use disorder were recruited from the Addiction Center at MUSC. This study was approved by MUSC institutional review boards. All participants provided written informed consent. The cannabis smoking cohort included 16 cannabis smokers and 27 non-smoking controls. The cannabis smokers were on current non-injection cannabis use but not on prescription drug and other illicit drug uses identified by chart reviews and urine tests. The clinical characteristics of cannabis smokers were shown in Supplementary Table 1. We conducted a Timeline Follow-back method, a web-based self-administered, to assess frequency and quantity of past 90-day cannabis uses prior to the study visit. Whether the participants used cannabis and the number of joints, blunts, pipes, bowls, vaporizers, spliffs, edibles, or other methods used. If participants shared a joint/blunt/etc. or otherwise did not use a full joint/blunt/etc., partial numbers were reported. Daily gram calculations of THC uses were calculated .Non-smoking controls were recruited from the Medical University of South Carolina and University of Connecticut by advertisement on the campus and chosen by self-report of non-drug use. Cannabis-smoking individuals with cannabis use disorder were recruited from the Addiction Center at MUSC. This study was approved by MUSC institutional review boards. All participants provided written informed consent. The cannabis smoking cohort included 16 cannabis smokers and 27 non-smoking controls. The cannabis smokers were on current non-injection cannabis use but not on prescription drug and other illicit drug uses identified by chart reviews and urine tests. The clinical characteristics of cannabis smokers were shown in Supplementary Table 1.
We conducted a Timeline Follow-back method, a web-based self-administered, to assess frequency and quantity of past 90-day cannabis uses prior to the study visit. Whether the participants used cannabis and the number of joints, blunts, pipes, bowls, vaporizers, spliffs, edibles, or other methods used. If participants shared a joint/blunt/etc. or otherwise did not use a full joint/blunt/etc., partial numbers were reported. Daily gram calculations of THC uses were calculated .For individuals with cannabis use disorder, we assessed exclusionary psychiatric diagnoses using appropriate modules of the MiniInternational Neuropsychiatric Interview, as described in our previous study. Briefly, The M.I.N.I., a brief structured interview, was to assess current Diagnostic and Statistical Manual of Mental Disorders, 5th edition diagnoses. Because the M.I.N.I. only assesses current diagnoses and a more thorough history of substance use is needed, the substance use module of the Structured Clinical Interview for DSM-V was used for substance use disorder diagnosis. Drug screens were performed using the onTrak test cup, an in vitro diagnostic test for the qualitative detection of drug or drug metabolite in the urine. Results of urine screenings were used to substantiate self-reports of cannabis use.The saliva micro-biome was analyzed and compared between cannabis smoking individuals and non-smoking control individuals. We found that cannabis smoking was associated with decreased oral microbial diversity compared to those in the non-smoking control group. The oral microbial communities differed in the two study groups reflected by the b-diversity. Next, we examined the relative abundance of individual bacterial taxa. The phylum Proteobacteria were decreased in cannabis smokers compared with those of non-smoking controls . At the genus level, enrichment of Neisseria was lower in cannabis smokers than in non-smokers; in contrast, Actinomyces, Veillonella, Megasphaera, and Streptococcus were found to increase among cannabis smokers .
At the species level, 36 species were significantly different in cannabis smokers compared to non-smoking controls after adjusting for multiple comparisons , including 16 species that were decreased and 20 species that were increased in cannabis smokers compared to non-smoking controls . Among the taxa that were increased in the saliva of cannabis smokers, five belonged to the Streptococcus genus, and four belonged to the Actinomyces genus. Among the taxa that were enriched in the saliva of non-smoking controls, six belonged to the Neisseria genus . No differences were observed based on quantity of cannabis use or presence of neurological disease history between the heavy users and light users , and between smoked cannabis containing THC and cannabis containing no THC . Nonetheless, the enrichment of A. meyeri was inversely correlated with the age of first cannabis use . To study which taxa may represent the cannabis smoking oral micro-biome, we reanalyzed and compared the saliva micro-biome from tobacco smokers and non-smoker controls from our published data. Consistent with the results from previously published studies, we found increased Streptococcus and decreased Neisseria in the oral micro-biome of tobacco smokers compared with those in non-smoking controls , which was similar to cannabis smokers. However, Actinomyces genus was only increased in cannabis smokers but not in tobacco smokers . To further analyze Actinomyces genus bacteria, we have shown four Actinomyces species bacteria that were significantly increased in cannabis smokers . Only Actinomyces turicensis was increased in tobacco smokers when compared with non-smoker controls . We further analyzed the difference after adjusting for age, sex, and/or alcohol use. All differences between cannabis users and controls identified in the univariate analysis shown in Fig. S1 remained significant after adjusting for sex, age, and alcohol consumption, although P values were attenuated slightly . When comparing tobacco users to controls, only Streptococcus and A. turicensis , but not Neisseria, remained significant after adjusting for sex and age.
Cannabis smokers have shown premalignant lesions in the oral mucosa with surface decay relative to a control group. The smoking and altered micro-biome composition may lead to a compromised mucosal epithelial barrier, which results in the translocation of bacteria or microbial products into circulation. Thus, bacterial fragments or whole bacteria can appear in the blood from translocation and thereby influence the immune system. To study oral microbial translocation in cannabis users, we evaluated the plasma levels of IgG antibody against antigens derived from A. meyeri, A. odontolyticus, and N. elongata. Plasma levels of IgGs against A. meyeri antigens tended to increase in the cannabis smokers compared to controls , while similar levels of IgGs against antigens from the other two bacteria were observed . These results imply that A. meyeri or its antigens may preferentially translocate from the oral mucosa to the circulation in the setting of an altered oral or periodontal environment in cannabis smokers.In a previous study, oral administration of Campylobacter jejuni activated state of neurons in nucleus tractus solitarius and increased c-Fos expression in the hypothalamic paraventricular nucleusas in mice. To determine if cannabis use-associated oral micro-biome affects CNS, we inoculated live A. meyeri, A. odontolyticus, and N. elongata into the oral cavity of C57BL/6 mice. A. meyeri and A. odontolyticus are oral commensal bacteria and were enriched in the oral micro-biome of cannabis smokers found in this study. N. elongata, which was enriched in non-smoking controls , was used as a control. We examined mouse activity through a uniformly cylindrical arena. The behaviors of mice were quantified and shown by global activity, total distance traveled , average speed , and resting time. The behavior of N. elongata-treated mice in the arena was comparable with the PBS-treated mice. However, compared with N. elongata-treated mice, A. meyeri-treated mice exhibited decreases in global activity,trim bin tray distance traveled, and mean speed, as well as increases in resting time . Next, we have evaluated amyloid production in mouse brain tissues as it is a marker of neurodegenerative diseases. Although Ab 40 tended to be increased in A. meyeri-treated mice, there was no statistical difference between any two groups .
Notably, the Ab 42 peptide in the brain from A. meyeri-treated mice was increased significantly compared to the control groups . To validate alterations in oral and gut micro-biome, we collected the samples from oral swab and stool one week after the final oral administration of live bacteria. specific bacteria were quantified using qPCR, and the abundance of each bacterium was normalized by total 16S rDNA. We con- firmed oral inoculated bacteria by qPCR . Notably, both A. odontolyticus and N. elongata, but not A. meyeri, presented in the oral swab of some mice from the PBS group, suggesting A. odontolyticus and N. elongata may be oral commensal micro-biome in mice . A. meyeri did not present in any group except the orally inoculated A. meyeri group. Furthermore, N. elongata presented in the stool samples from some mice after oral inoculation, but was not signififi- cantly elevated compared to the controls; elevation of A. meyeri in stool after oral inoculation was extremely limited . These results indicate that A. meyeri may not be an oral commensal bacterium in B6 mice, but it can colonize well on the surface of the oral cavity during oral inoculation.Long-term heightened systemic inflammation affects neuroin- flammation, and neuroinflammation may result in CNS damage and Ab protein production. To evaluate systemic inflammation after6-month oral bacterial inoculation, we tested plasma levels of 33 cytokines or chemokines in mice. Among these inflammatory markers, only MIP-1a and TNF-a levels were increased in the A. meyeri group compared to the control group . Plasma levels of MIP-1a were also increased in the A. odontolyticus group compared to the control N. elongata group . However, blood levels of the other microbial TLR-downstream proinflammatory cytokines including IL-1b, IL-6, IFN-g, IP-10, MCP-1, and MIP-2 were similar among the four study groups.We analyzed the correlation of A. meyeri, A. odontolyticus, and N. elongata with cytokine levels, and found that N. elongata enrichment in the saliva was directly correlated with levels of IFN-g in the cannabis smokers. However, after FDR adjustment, the p value was not significant.
No correlation was found between plasma cytokine levels and saliva enrichment of A. meyeri or A. odontolyticus. Infiltrating monocyte-derived macrophages in the CNS was related to the progression of neurodegenerative disease pathology. To investigate the potential mechanism of oral A. meyeri-mediated CNS abnormalities, we evaluated myeloid cell migration and infiltration by IHC using mouse brain tissues after oral inoculation of bacteria. Notably, macrophage infiltration was increased in the brain of A. meyeri-treated mice when compared to N. elongata or PBS-treated mice . In in vitro studies, A. meyeri, A. odontolyticus, and LPS significantly enhanced cell transmigration through microporous membranes in human macrophages via the MyD88 cell signaling pathway . Phagocytosis is a major function of antigen-presenting cells. Bacterial products activate antigen-presenting cells and decrease phagocytic capacity. We found that treatment of N. elongata significantly decreased phagocytosis in both human primary monocyte-derived macrophages and THP-1-derived macrophages. However, treatment of Actinomyces species bacteria maintained macrophage phagocytic capacity similar to those of unstimulated cells . Moreover, as expected, A. odontolyticus or N. elongata induced TNF-a, IL-1b, and IL-6 production in human primary monocytes . Unexpectedly, A. meyeri did not activate monocytes to produce TNF-a, IL-1b, and IL-6 .Long-term cannabis users may suffer disturbed brain connectivity, cognitive impairment, and psychological disorders, but the exact mechanisms are not fully understood. In the current study, we found that chronic cannabis use correlated with alterations of several taxa of the oral micro-biome.