Date: Jan 05, 2021 Author: Christine L. Foxx1,2, Jared D. Heinze1,2, Antonio González3, Fernando Vargas4,5, Michael V. Baratta6 Source: Frontiers in Physiology (
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Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day --28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days --21, --14, --7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0--56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days --1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.
Introduction
Stress-related psychiatric disorders, such as major depression, affect more than 17.3 million adults aged 18 and older in the United States each year (Substance Abuse, and Mental Health and Services Administration, 2018). One important risk factor for the development of stress-related psychiatric disorders, including major depressive disorder and anxiety disorders, is psychosocial stress (Fan et al., 2015). Despite evidence supporting the associations between psychosocial stress, depression, and anxiety, the biological mechanisms by which psychosocial stress and stress-related psychiatric disorders such as depression are related to one another are poorly understood. Studies suggest that chronic low-grade inflammation, particularly in response to lower subjective social status, may be an important factor with a causal role in the development of depression (Bellingrath et al., 2010; Rohleder, 2014; Eddy et al., 2016). In fact, multiple convergent lines of evidence in humans and in animal models suggest that exaggerated or inappropriate peripheral inflammation increases the risk of stress-related psychiatric disorders (Hodes et al., 2014; Miller and Raison, 2016). While a number of factors contribute to individual variability in peripheral proinflammatory immune responses, the microbiome has recently received considerable attention (Cryan and Dinan, 2012; Belkaid and Hand, 2014; Lowry et al., 2016; Flux and Lowry, 2019).
Throughout evolution, humans have coevolved with diverse microorganisms, including prokaryotes, eukaryotes, and viruses, which together constitute the human microbiome (Hooper et al., 2002; Dave et al., 2012). Specific microorganisms have been shown to prime immunoregulatory circuits and suppress pathological inflammation through the actions of regulatory T cells (Treg) (Rook et al., 2004). These include: (1) commensal microbiota that have been altered by the Western lifestyle (von Hertzen et al., 2015; Sonnenburg and Sonnenburg, 2019); (2) pathogens associated with "old infections" from the hunter-gatherer period of human evolution, including Paleolithic strains of Mycobacterium tuberculosis that were less pathogenic than extant ones (Comas et al., 2013), helminths including gut parasites (Babu et al., 2006), and Helicobacter pylori (Atherton and Blaser, 2009; reviewed in Rook, 2010; Rook et al., 2014); and (3) organisms from the natural environment, such as water- and soil-associated saprophytes including Mycobacterium vaccae, which were frequently encountered by humans and tolerated by the immune system (Rook et al., 2004). However, the human microbiome may have shifted radically as a result of living a modern urban lifestyle, with entire classes of these microorganisms that prime immunoregulatory circuits either reduced or absent (Rook et al., 2004; Blaser and Falkow, 2009; von Hertzen et al., 2011; Sonnenburg and Sonnenburg, 2019). Compositional alterations in the microbiome due to modern urban living are thought to alter the manner in which the peripheral immune system responds to challenge, resulting in a deficiency of Treg development, a shift toward imbalanced immunoregulation, chronic low-grade inflammation (Rook et al., 2004), and exaggerated proinflammatory responses to psychosocial stressors (Böbel et al., 2018).
Consistent with the "Old Friends" hypothesis, individuals raised in an urban environment have an increased proinflammatory immune response to psychosocial stress relative to individuals raised in a rural environment with farm animals (Böbel et al., 2018). Although many factors have been identified that may contribute to the industrialized microbiota (Sonnenburg and Sonnenburg, 2019), chronic low-grade inflammation (Rohleder, 2014), and vulnerability to exaggerated or prolonged inflammatory responses to psychosocial stress (Böbel et al., 2018), one common factor associated with the modern lifestyle is circadian disruption. Circadian disruption alters the microbiome (Voigt et al., 2014, 2016; Deaver et al., 2018; Wu et al., 2018; Parkar et al., 2019), induces chronic low-grade inflammation (Li et al., 2018; Inokawa et al., 2020), and induces exaggerated inflammatory responses to immune challenge (Castanon-Cervantes et al., 2010; Adams et al., 2013; Brager et al., 2013; Guerrero-Vargas et al., 2015; Comas et al., 2017). Circadian disruption, in turn, increases risk of stress-related psychiatric disorders (Karatsoreos, 2014). This reasoning has led to the development of strategies to promote stress resilience and to prevent or treat psychiatric disorders by restoring some of the lost "Old Friends" through microbial-based interventions. Repeated immunization with Mycobacterium vaccae NCTC 11659 (M. vaccae), one such microbial-based intervention, has been shown to shift immune signaling toward an immunoregulatory phenotype and prevent inappropriate inflammation (Lowry et al., 2016; Reber et al., 2016b; Fonken et al., 2018; Frank et al., 2018; Amoroso et al., 2019a,b; Loupy et al., 2019).
Here we evaluated the effects of immunization with M. vaccae NCTC 11659, a soil-derived bacterium with anti-inflammatory and immunoregulatory properties (Zuany-Amorim et al., 2002b), in a "two hit" model of stress exposure, incorporating stressors that are common in modern urban lifestyles such as circadian disruption and psychosocial stress. We evaluated the effects of M. vaccae immunization and chronic disruption of rhythms (CDR) on behavioral responses during acute social defeat (SD) that have been identified as determinants of individual variability in stress resilience and vulnerability to anxiety- and depressive-like behavioral responses (Koolhaas et al., 1999; Veenema et al., 2007; Wood et al., 2010; Wood and Bhatnagar, 2015). In addition, we assessed the effects of M. vaccae, CDR, and SD on subsequent cognitive performance in the object location memory (OLM) test. Finally, we evaluated potential mediators and moderators of the effects of M. vaccae, CDR, and SD, including measures of alpha and beta diversity in the gut microbiome, the fecal metabolome, the host plasma metabolome, and serotonergic gene expression in the midbrain and pontine raphe complex, brain systems implicated in individual variability in vulnerability to anxiety- and depressive-like behavioral responses (Wood et al., 2015; Hassell et al., 2018).
