The contribution of microglia to “immunization against stress”

Graham Rook and Christopher Lowry have been studying Mycobacterium vaccae (M. vaccae) for quite some time. Let’s say it all started with dirt, or more specifically, the benefits of some kinds of dirt as put forth in the “old friends” hypothesis. The hypothesis posits that early-life exposure to diverse macro- and microorganisms and microbiota that accompanied mammalian evolution tunes and primes our immune systems to withstand later insults (Rook et al., 2013). One of the “old friends” is M. vaccae, a nonpathogenic species of the Mycobacteriaceae family of bacteria that lives naturally in soil. Research has shown that these bacteria may be beneficial by programming the adaptive immune system. Animals immunized (by spaced subcutaneous injections) with heat-killed M. vaccae showed altered immune responses, with a skewing of the T helper lymphocyte profile towards Th1 (IFNγ producing) (Janssen et al., 2001) and away from Th2 (IL-5 producing) (Wang and Rook 1998). Consequently, M. vaccae immunization has been tested as an immunotherapy for a number of diseases such as allergic asthma, cancer, and tuberculosis. Now it is being considered as a treatment for depression. Lowry’s team in 2016 found that immunization of mice with M. vaccae prior to exposure to chronic psychosocial housing stress reduced stress-induced development of both colitis and anxiety via skewing of the adaptive immune system towards regulatory T (Treg) cells. Treg cells in turn produce anti-inflammatory cytokines, specifically, IL-10 and TGFβ (Reber et al., 2016). These striking findings attracted media attention for a potential therapeutic “immunization against stress” and made the top 10 list for breakthroughs by the Brain and Behavior Research Foundation that year.

The newest study by the Rook/Lowry team in collaboration with the Frank/Maier/Watkins team (Frank et al., 2018a) shows that immunization-induced changes in the peripheral immune system can translate to changes in the CNS via alterations in the microglial compartment. Their previous work indicated that following immunization, serotonin markers were elevated in the dorsal raphe nucleus, and microglia in the medial prefrontal cortex were more densely immunostained with the antibody Iba1 (Reber et al., 2016). However, little was known about how the peripheral immune signals were transmitted to brain and whether a central immune response was involved. Frank et al. (2018a) have now contributed important new insights to these mechanisms. In searching for suspects, the authors found that M. vaccae immunization elevated expression levels of Il4, Cd200r1, and Mrc1 mRNA and IL-4 protein while reducing levels of Nlrp3 (a key component of the inflammasome) and Nfkbia (IκBα, the rate-limiting molecule in NF-κB signaling) mRNA in the hippocampus. In general, the elevated transcripts are anti-inflammatory, and the reduced transcripts are pro-inflammatory. The cellular origin of these signals is not known, but microglia are a likely candidate.

The acute stressor used in this study—exposure to a single session of 100 inescapable footshocks—decreased levels of Cd200 and increased levels of high-mobility group box 1 (Hmgb1) mRNA. These two genes are candidates that might translate stress effects onto altered microglial activity (Deczkowska et al., 2018, Weber et al., 2015). As predicted, prior M. vaccae immunization blunted those effects. The inescapable shock session primed the microglia as determined ex vivo by LPS challenge to isolated hippocampal microglia. Prior immunization blunted the priming of gene expression of Il1b and Nfkbia. Finally, as before, immunization blocked the stress-induced increases in anxiety behavior measured by the juvenile social exploration test.

Priming is a useful concept that describes the behavior of microglia in response to a new challenge after having been exposed to earlier challenges. Primed microglia may appear morphologically normal following the initial trigger—e.g., stress—and may only show their priming effect following a subsequent challenge—e.g., LPS stimulation. The authors argued that HMGB1, whose levels are affected by disrupted CD200:CD200R1 signaling (Frank et al., 2018b), is the major molecular mechanism that underlies microglial priming. A wide range of stressors can prime microglia (Calcia et al., 2016), and the generalizability of these data to other paradigms needs to be assessed.

It is somewhat puzzling that the effects of M. vaccae vaccination on gene expression found in the hippocampus were not found in the amygdala, as both regions are known to be involved in fear responses associated with inescapable shock. The authors removed the adjacent choroid plexus prior to removal of hippocampus to rule out a possible contaminating source of hippocampal immune gene expression. The choroid plexus and meninges are the predominant locations of peripheral immune cells in the brain (Korin et al., 2017) and may be key waystations by which the altered peripheral immune system imparts changes onto the CNS (Herkenham and Kigar, 2017). Signaling across the blood-brain barrier and mechanisms conferring regional specificity of action remain missing components to this story. Some of the potential pathways are discussed by Frank et al. (2018a), including the possibility that T cells might migrate from the meningeal compartment into the hippocampus.

The study points to microglia and HMGB1 as key mediators of the stress effects. What would induce microglial activation, reduction of CD200R1 levels, and production of alarmins such as HMGB1? One candidate, stress-induced glucocorticoids (Frank et al., 2015, Singhal and Baune, 2017), appeared to be ruled out by the data. Oxidative stress is another candidate that merits future study (Tang et al., 2011). Finally, microbiota, the composition of which might be altered by both stress and immunization or probiotic treatment, may play a role (Erny et al., 2015, Singhal and Baune, 2017) and could bring us full circle back to the “old friends” hypothesis, as supported by the group’s recent work (Bobel et al., 2018). This intriguing possibility deserves further study.