
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38252816
202320425
10.1073/pnas.2320425121
commCommentaryneuroNeuroscience424
437
Commentary
Biological Sciences
Neuroscience
Gut microbiota: A key susceptibility factor in social anxiety disorder
Diaz Heijtz Rochellys rochellys.heijtz@ki.se
a 1 https://orcid.org/0000-0001-6821-2569

aDepartment of Neuroscience, Karolinska Institutet, Stockholm 171 77, Sweden
1Email: rochellys.heijtz@ki.se.
22 1 2024
30 1 2024
22 7 2024
121 5 e2320425121Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Vetenskapsrådet (VR) 501100004359 2018-06232 Rochellys Diaz-Heijtz Hjärnfonden (Brain Foundation) 501100003792 FO2022-0199 Rochellys Diaz-Heijtz Olle Engkvists Stiftelse (Stiftelsen Olle Engkvist Byggmästare) 501100004200 226-0123-REK-0002 Rochellys Diaz-Heijtz
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pmcSocial anxiety disorder (SAD), also known as social phobia, is a prevalent psychiatric condition characterized by intense fear or anxiety associated with being scrutinized in one or more social situations (1). The onset of the disorder typically occurs in early life, with the highest incidence between the ages of 10 and 19 and often follows a chronic lifelong trajectory (2). However, the causes and pathogenesis of SAD are still poorly understood (3). In recent years, there has been a growing interest in the potential role of the gut microbiota—the trillions of microorganisms that colonize our gastrointestinal (GI) tract—in the pathogenesis of complex human brain disorders, including psychiatric disorders (4). The gut microbiota is now acknowledged as an important modulator of brain development, function, and behavior. Recent data have implicated the gut microbiota in social processes across the animal kingdom (5). Interestingly, a recent study has demonstrated that individuals with SAD exhibit a distinct microbiota composition compared to age-matched neurotypical individuals (6), implicating the gut microbiota as a potential susceptibility factor in SAD.

In PNAS, Ritz et al. (7) investigate the causal link between the gut microbiota and host social fear responses by transplanting fecal microbiota from patients with social anxiety disorder (SAD) into mice with an antibiotic-depleted microbiota (Fig. 1). The authors selected six individuals affected by SAD and six healthy neurotypical individuals from a well-characterized cohort based on various key host parameters and metrics (such as microbiota composition, Liebowitz Social Anxiety Scale scores, body mass index, and sex) to serve as microbiota donors for the fecal microbiota transplantation (FMT) studies in mice. Importantly, individuals exposed to medication known to impact the gut microbiota composition, such as psychotropic drugs, antibiotics, and microbiota-focused interventions (prebiotics, probiotics, etc.), were excluded as microbiota donors. Following the specific donor FMT (SAD vs. Neurotypical), mice were assessed for social fear, sociability, social cognition, and stress-coping behaviors.

Fig. 1. Transfer of fecal microbiota from patients with SAD into mice leads to an increased sensitivity to social fear.

Ritz et al. (7) demonstrate that the transfer of fecal microbiota from patients with SAD into mice leads to a heightened sensitivity to social fear, using a behavioral test specifically designed to model social anxiety in rodents (8). These effects are specific to social fear, as there were no differences between groups in sociability or social novelty preferences as measured by the three-chambered approach test or other tests relating to stress-coping behaviors. Consistent with these observations, recent studies have shown that a significant number of individuals affected by SAD report typical (or even higher) levels of extraversion and novelty-seeking (openness to new experiences), despite their anxiety related to social situations (9, 10). SAD indeed appears to be multidimensional, potentially aligning with the conceptual framework of a spectrum disorder.

The hypothalamic–pituitary–adrenal (HPA) axis regulates behavioral responses to stressors in both humans and animals and is dysregulated in a wide range of psychiatric disorders, including major depression and general anxiety disorder (often comorbid with SAD). While not consistently observed (11), some studies have found evidence supporting hypo-responsiveness of the HPA-axis in SAD (12). In this study, Ritz et al. (7) demonstrate that mice receiving FMT from individuals with SAD exhibit lower levels of basal corticosterone (the primary adrenal corticosteroid in rodents) when compared to mice receiving FMT from neurotypicals. Notably, there was no observed difference in corticosterone levels following stress. It would be interesting to further explore the relationship between HPA axis activity, both under basal and stress conditions and distinct personality subtypes of SAD (9, 10).

In summary, the study by Ritz et al. provides the first experimental evidence that the SAD microbiota induces behavioral changes relevant to this disorder, such as increased social fear.

In the cohort of SAD patients who served as microbiota donors, a significant reduction in circulating interleukin-10 (IL-10) was observed compared to the neurotypical group (albeit without any alterations in circulating pro-inflammatory cytokines) (7, 13). IL-10 is a pleiotropic cytokine that plays a crucial role in limiting host immune responses and maintaining normal tissue homeostasis. It functions primarily as an anti-inflammatory cytokine, but it can also exhibit immunostimulatory activities under specific conditions. Indeed, some reports have shown that individuals with immune-mediated inflammatory diseases such as multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis have a relatively high prevalence of SAD, exceeding that reported for the general population (14), suggesting a common role of inflammation and/or shared genetic factors. Surprisingly, the possible role of inflammation in the neurobiology of SAD has received little attention.

To assess the immune function of T cells within the gut of mice that received SAD FMT, Ritz et al. (7) conducted experiments where they stimulated gut explants, specifically the ileum (the final portion of the small intestine), with various compounds. These compounds were chosen to mimic the signals T cells would receive during natural immune responses, including lipopolysaccharide (LPS; a crucial outer membrane component of gram-negative bacteria), concanavalin A (ConA; a plant lectin known for its ability to activate T cells and natural killer cells), and T cell receptor clusters of differentiation 3 (CD3) and CD28. Fascinatingly, mice that received FMT from individuals with SAD showed reduced IL-17A, a pro-inflammatory cytokine, in response to stimulation by LPS and ConA, and a similar trend after CD3/CD28 stimulation and vehicle control. In the gut, IL-17A is mainly produced by T helper 17 (Th17) cells, a distinct lineage of CD4+ T cells that plays a crucial role in the immune response. During infection, IL-17A is a key cytokine that links T cell activation to neutrophil mobilization and activation (15). These observations suggest that SAD microbiota dampens gut immunity.

Mesenteric lymph nodes (MLNs) play a crucial role in T cell activation in both the small intestine and colon. Dendritic cells (DCs), acting as antigen-presenting cells, consistently migrate from intestinal tissues to the MLNs. There, they present antigens to lymphocytes, initiating and regulating the adaptive immune response. Ritz et al. (7) show that MLNs are particularly sensitive to modulation by FMT from patients with SAD. For instance, recipient mice of SAD FMT display a significant reduction in F4/80+ macrophages in the MLNs, along with a trend toward an increase in the relative number of CD4+ T helper cells. Additionally, a reduction of CD44 receptors on circulating CD4+ T helper cells was noted in the SAD group. CD44 is a prominent activation marker for T cell activation and is characteristic of long-lived memory cells (16). Furthermore, it enhances T cell receptor signaling by facilitating the delivery of lymphocyte-specific protein kinase, crucial for the development of IL-17-producing Th17 cells. Together, these data indicate that the microbiota from SAD patients strongly impacts peripheral immunity, particularly the IL-17A signaling pathway.

Next, Ritz et al. (7) investigated alterations in central immunity following SAD FMT, focusing on the corticolimbic circuitry—a complex network of cortical and limbic structures, including the medial prefrontal cortex (mPFC), bed nucleus of the stria terminalis (BNST), and medial amygdala (MeA). This circuitry plays a crucial role in regulating emotional processes and behaviors and is particularly sensitive to signals from the microbiota (17). In line with previous studies linking the gut microbiota to corticolimbic circuit structure and function, Ritz et al. demonstrated that brain’s specific expression of various neuroinflammatory-related genes was affected after SAD FMT. Specifically, arginase 1 (Arg 1), a marker of cellular repair activation following neuroinflammation, was significantly reduced in the mPFC, BNST, and MeA, while the expression of tumor necrosis alpha (Tnf-α), IL-10, and chemokine C-X-C ligand (Cxcl15) was significantly reduced within the MeA. Additionally, toll-like receptor 4 (Tlr4), implicated in inflammatory responses following LPS activation, was found to be reduced in the mPFC. Notably, Tlr4 knockout mice exhibit increased anxiety-like behavior and reduced social interaction (18). Both Arg1 and Tlr4 are primarily expressed by microglia, the brain’s resident macrophages, suggesting that the SAD microbiota influences microglial gene expression and function. Recent studies have uncovered the microbiota's impact on social behavior by stimulating microglial remodeling of forebrain circuits during early neurodevelopment (19). While neuronal circuit remodeling is less pronounced in adulthood, it is crucial to recognize that microglia continue to regulate the population of newly generated neurons in adults and actively contribute to synaptic remodeling (20). Therefore, it is essential to explore whether the SAD microbiota induces structural changes (e.g., dendritic morphology of pyramidal neurons in the MeA and PFC) by directly modulating microglial-specific cell populations within the corticolimbic circuitry and its potential relationship to social fear responses.

The neuropeptide oxytocin is a key modulator of the corticolimbic circuitry and plays a crucial role in diverse social behaviors, including stress-induced defensive and social coping responses (21). In their study, Ritz et al. (7) demonstrated that the SAD microbiota selectively reduces neuronal oxytocin in the BNST and various oxytocin-related markers (such as oxytocin receptor and vasopressin receptor 1b) in the MeA and PFC, indicating that the oxytocin system is particularly vulnerable to SAD microbiota-mediated signaling molecules to the brain.

In summary, the study by Ritz et al. (7) provides the first experimental evidence that the SAD microbiota induces behavioral changes relevant to this disorder, such as increased social fear. These changes are associated with impaired peripheral immune activation and disturbances within the corticolimbic circuitry implicated in SAD, identifying the gut microbiota as a potential therapeutic target. These findings also raise several questions: Which microbial-derived metabolites and/or microbial components are involved in transferring behavioral traits relevant to SAD? Are there sex- and age-dependent effects of SAD microbiota? Are specific brain circuits and cells more sensitive to modulation by the SAD microbiota?

R.D.-H.’s research is supported by grants from the Swedish Research Council, the Swedish Brain Foundation, and the Olle Engkvist Foundation.

Author contributions

R.D.H. wrote the paper and made the figure.

Competing interests

The author declares no competing interest.

See companion article, “Social anxiety disorder-associated gut microbiota increases social fear,” 10.1073/pnas.2308706120.
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