A novel soil-derived Mycolicibacterium decreases anxiety-like defensive behavioral responses in association with decreases in biomarkers of neuroinflammation and hippocampal microglial priming in adult male rats

Abstract

Major traumatic life events are risk factors for stress-related neuropsychiatric disorders, often accompanied by systemic inflammation, neuroinflammation, and microglial priming. As systemic inflammation, neuroinflammation, and microglial priming are considered risk factors for developing stress-related psychiatric disorders, one novel therapeutic strategy is to identify interventions that mitigate these responses. In this study, we investigated the effects of a novel soil-derived Mycolicibacterium, Mycolicibacterium sp. strain KGA-10, on in vitro immunoregulatory potential in murine bone marrow-derived dendritic cells (BMDCs) and on biomarkers of systemic inflammation, biomarkers of hippocampal neuroinflammation and microglial priming, and anxiety-like defensive behavioral responses in adult male rats exposed to inescapable tail-shock stress (IS). In Experiments 1, 2, and 3, BMDCs were exposed to the type strain, Mycolicibacterium vaccae ATCC 15483 (0, 10, 30, 100, 300 μg/mL; Experiment 1) or M. sp. strain KGA-10 (100 μg/mL; Experiments 2 and 3) or sterile borate-buffered saline (BBS) vehicle followed, 24 h later, by exposure to lipopolysaccharide (LPS; 250 ng/mL) or a cell culture media vehicle, then, 24 h later, assessed for Il10, Il12a, and Il12b mRNA expression. Exposure of murine BMDCs to M. vaccae ATCC 15483 or M. sp. strain KGA-10 induced an immunoregulatory phenotype, characterized by increased ratios of Il10:Il12a and Il10:Il12b mRNA expression in both naïve and lipopolysaccharide- (LPS; 250 ng/mL) challenged conditions. In Experiment 3, adult male rats received weekly injections of heat-killed M. sp. strain KGA-10 (0.1 mg/0.1 mL, s.c.) or sterile BBS vehicle over three weeks prior to IS. Anxiety-like defensive behavioral responses were assessed 24 h following IS or home cage control conditions using the juvenile social exploration (JSE) test, while biomarkers of hippocampal neuroinflammation and microglial priming were assessed using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). M. sp. strain KGA-10 treatment promoted an anti-inflammatory immunophenotype, evidenced by decreased hippocampal Il12a, and decreased biomarkers of microglial priming, Nfkbia and Nlrp3 mRNA expression among rats exposed to IS, in association with prevention of IS-induced increases in anxiety-like defensive responses in the JSE test. These findings suggest that M. sp. strain KGA-10 is a promising candidate for a novel intervention for promotion of stress resilience and prevention of stress-related psychiatric disorders.

Graphical abstract

Introduction

Stress-related psychiatric disorders, including anxiety disorders, mood disorders, and trauma and stressor-related disorders, such as posttraumatic stress disorder (PTSD), are increasing globally (Brewin et al., 2025; Fan et al., 2025; World Health Organization, 2025). The increases in the prevalence of stress-related psychiatric disorders are associated with increases in chronic low-grade inflammation, particularly in persons living urban, industrialized lifestyles (Franck et al., 2025; Hidaka, 2012; McDade et al., 2012). Chronic-low grade inflammation, neuroinflammation, and microglial priming in turn are thought to be risk factors for development of stress-related psychiatric disorders (Eraly et al., 2014; Gao et al., 2018; Katrinli et al., 2022; Rohleder, 2014; Schultebraucks et al., 2021; Tay et al., 2017). One mechanism that may be contributing to chronic low-grade inflammation in those living in modern urban societies is reduced exposures to diverse microbial environments, which have the ability to modulate stress-related behavioral responses through the regulation of metabolic and inflammatory signaling (Cryan et al., 2020; Lowry et al., 2016). The “hygiene hypothesis” (Rook, 2007), the “Old Friends” hypothesis (Rook et al., 2004; Rook, 2007; Rook, 2023; Rook et al., 2013), the “Farm Effect” (von Mutius, 2022), the biodiversity hypothesis (Rook, 2023; Rook et al., 2013), the “industrialized microbiota hypothesis” (Sonnenburg & Sonnenburg, 2019), and the disappearing microbiota hypothesis (Blaser, 2015; Blaser & Falkow, 2009) have in common the feature of reduced exposure to diverse microbial environments, which is particularly evident in urbanized societies, leading to immune dysregulation and increased risk of inflammatory diseases as well as stress-related psychiatric disorders, in which inflammation is considered a risk factor (Flux & Lowry, 2023).

The “Old Friends” consist of microorganisms with which mammals coevolved, including: (i) the commensal microbiota, which have been altered by the Western lifestyle, including a high-fat, high-sugar, Western-style diet that is commonly low in fiber (Sonnenburg & Sonnenburg, 2014; von Hertzen et al., 2015); (ii), pathogens associated with the “old infections” that were present throughout life in evolving human hunter-gatherer populations (Atherton & Blaser, 2009); and (iii) organisms from the natural environment with which humans were inevitably in daily contact (and so had to be tolerated by the immune system), including environmental saprophytes (Rook et al., 2014). Exposure to microbial “Old Friends” is essential to provide adequate immunoregulation, to prevent inappropriate inflammation, and to promote a stress resilient behavioral phenotype (Böbel et al., 2018; Langgartner et al., 2019; Langgartner et al., 2025; Lowry et al., 2016; Ohnmacht et al., 2015; Rook et al., 2014; Rook et al., 2013; Sefik et al., 2015).

Previously, our lab has investigated the stress resilience effects of subcutaneous injection of select “Old Friends”. These previous studies have shown that subcutaneous (s.c.) injection of Mycolicibacterium vaccae NCTC 11659 or M. vaccae ATCC 15483 is associated with: 1) suppression of stress-induced systemic inflammation (Langgartner et al., 2023; Reber et al., 2016a; Reber et al., 2016b); 2) prevention of stress-induced hippocampal neuroinflammation (Loupy et al., 2021); 3) reduction in biomarkers of microglial priming (e.g., Nfkbia, Nlrp3, and Hmgb1); (Desmond et al., 2025; Fonken et al., 2018a; Frank et al., 2018; Noronha et al., 2022), and attenuation of stress-induced microglial priming (Fonken et al., 2018c; Frank et al., 2018; Sanchez et al., 2022); and 4) promotion of anxiolytic and stress-resilient behavioral phenotypes (Amoroso et al., 2020; Amoroso et al., 2019; Bowers et al., 2021; Desmond et al., 2025; Foxx et al., 2020; Frank et al., 2018; Loupy et al., 2021; Reber et al., 2016b). These findings provide strong evidence that soil-derived mycobacteria such as M. vaccae NCTC 11659 can prevent stress-associated inflammation, neuroinflammation, hippocampal microglial priming, and anxiety-like defensive behavioral responses. Thus, it is of interest to determine if this is a unique feature of M. vaccae strains, or if other strains of rapidly growing mycobacteria may also have these properties. The focus on isolating and testing new strains may enable the transition from general health-promoting bacteria to “precision psychobiotics” or “precision postbiotics”. Further, evaluating novel isolates against an established benchmark allows for the identification of enhanced efficacy, additional benefits, and optimal growth profiles of other species and strains within a genus.

Although the main focus for development of probiotics and postbiotics to advance human health has been on the commensal microbiota category of “Old Friends”, organisms from the natural environment with which humans were inevitably in daily contact, including environmental saprophytes such as mycolicibacteria, represent a largely unexplored resource. The soil ecosystem is incredibly diverse (Hug et al., 2016), offering a vast, largely untapped reservoir for discovering new strains with therapeutic potential, such as anti-inflammatory, immunoregulatory, and stress resilience properties. In addition, soil-based postbiotics may be safer for long-term use because they often do not have the same risk of transferring antibiotic resistance genes relative to probiotics (Thorakkattu et al., 2022).

In this study, we investigated the effects of a novel, soil-derived Mycolicibacterium, M. sp. strain KGA-10, on in vitro immunoregulatory potential in murine bone marrow-derived dendritic cells (BMDCs) and on in vivo hippocampal neuroinflammation, biomarkers of hippocampal microglial priming, and anxiety-like defensive behavioral responses. In this study we specifically chose the juvenile social exploration (JSE) test as it is sensitive to the (i) uncontrollability of the stressor (Christianson et al., 2008), (ii) classic anxiogenic and anxiolytic pharmacological treatments (Christianson et al., 2008), both supporting the conclusion that reduced social exploration by IS reflects a state of anxiety. Other classic anxiety tests (e.g., elevated plus-maze) are not sensitive to stressor controllability (Grahn et al., 1995). Since tissue was collected 24 h post-IS, we expected that markers of microglial priming, rather than neuroinflammation, would be impacted by IS. We hypothesized that M. sp. strain KGA-10 induces an anti-inflammatory and immunoregulatory immunophenotype in murine BMDCs in vitro, and, when given by s.c. injection, decreases biomarkers of hippocampal microglial priming, and prevents stress-induced anxiety-like defensive behavioral responses in the inescapable tail shock stress (IS)/juvenile social exploration (JSE) paradigm (a behavior sensitive to microglial priming and exaggerated neuroinflammatory processes) (Andersen et al., 2023; Goshen & Yirmiya, 2009; Weber et al., 2015).

2. Materials and Methods

2.1. Selection of animal models for in vitro BMDC assays and in vivo behavioral studies

Murine systems are used for in vitro experiments due to well-characterized immune cell models and reagents and extensive immunological research in mice. In contrast, all prior in vivo experiments evaluating the effects of administration of mycolicibacteria on inescapable shock-(IS-) induced neuroimmune and behavioral outcomes have been conducted in rats, and the current studies use the same species for consistency. It remains unclear whether the IS-induced neuroimmune phenomena examined here also occur in mice. This is a widely adopted approach that leverages the strength of each species for addressing specific experimental questions.

2.2. Experiment 1: Validation of an assay for immunoregulatory potential of rapidly growing mycobacteria: In vitro assay to evaluate the dose dependent effects of M. vaccae ATCC 15483 on the immunophenotype of murine BMDCs

In order to assess the immunoregulatory potential of M. vaccae ATCC 15483, we assessed the ability of M. vaccae ATCC 15483 to shift the immunophenotype of murine BMDCs toward an anti-inflammatory and immunoregulatory phenotype. Specifically, we evaluated the ability of M. vaccae ATCC 15483 to increase the ratios of Il10:Il12a and Il10:Il12b, as these ratios are thought to play an important role in programming DCs toward a regulatory DC (DCreg) phenotype, with potential to induce the differentiation of naïve T cells toward regulatory T cells (Treg) and away from effector T cells, such as T helper 1 (Th1) cells, Th2 cells, and Th17 cells, particularly in response to “Old Friends” (Arnold et al., 2012; Rook & Lowry, 2008).

2.2.1. Experiment 1: Animals

Thirty-five-day-old male C57BL/6N mice (Charles River, Raleigh, NC, USA) were used to extract bone marrow cells after one week of acclimation to the vivarium. The mice were maintained under a 12:12 h light/dark cycle with lights on at 7 a.m. The mice were pair-housed separately in Static Allentown micro isolator filter-topped caging (Allentown, Allentown, NJ, USA), cage model # PC75JHT (186 mm w x 298 mm l x 128 mm h), cage top: # MBT7115RH, containing 7090 Teklad Sani-Chip bedding (Envigo (now Inotiv), Madison, WI, USA) and enrichment in the form of a Nestlet (Ancare Corp, Bellmore, NY, USA) and crinkle paper (Shepherd Specialty Papers, Watertown, TN, USA). Mice were given ad libitum access to irradiated rodent chow (Teklad 2918; 18% protein, 6% fat, Envigo (now Inotiv, Madison, WI, USA) and reverse osmosis (R/O) water supplied by a Hydropac Advanced Lab Animal Watering System (Lab Products, Inc., Seaford, DE, USA). The research described here was compliant with the Guide for the Care and Use of Laboratory Animals, Eighth Edition (Institute for Laboratory Animal Research, The National Academies Press, Washington, D.C., 2011) and was approved by the University of Colorado Boulder Institutional Animal Care and Use Committee (IACUC). The research is reported in compliance with The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research (Percie du Sert et al., 2020). All efforts were made to minimize the number of animals used and their suffering.

2.2.2. Experiment 1. Experimental design: Effects of M. vaccae ATCC 15483 and LPS on cytokine mRNA expression in murine BMDCs

In order to screen soil-derived mycobacteria for potential for stress resilience properties, we first set out to establish a cell-based assay designed to determine the immunoregulatory potential of individual Mycolicibacterium strains, using the type strain M. vaccae ATCC 15483 as a reference strain. The overall experimental design for Experiment 1 is illustrated in Fig. 1. Briefly, Experiment 1 was a 5 x 2 factorial design: 5 (10 μg/mL, 30 μg/mL, 100 μg/mL, 300 μg/mL whole cell, heat-killed M. vaccae ATCC 15483 versus sterile borate-buffered saline (BBS) vehicle) x 2 (250 ng/mL LPS versus RPMI 1640 cell media vehicle). There were ten technical replicates (i.e., ten wells; n = 10) per treatment group. DIV 0 was defined as the day of bone marrow extraction (11/16/2020). On DIV 7, BMDCs were exposed to sterile BBS or 10 μg/mL, 30 μg/mL, 100 μg/mL, 300 μg/mL M. vaccae ATCC 15483. Stocks of M. vaccae ATCC 15483 were swirled each time before pipetting into the wells to ensure the suspension was distributed evenly. Concentrated M. vaccae ATCC 15483 stock solutions (100X) were made fresh every time for every concentration in sterile BBS. A total of 15 μl of 33.3X M. vaccae ATCC 15483 stocks or sterile BBS were pipetted on each well. On DIV 8, BMDCs, still exposed to prior experimental conditions, were additionally challenged with lipopolysaccharide (LPS; Escherichia coli 0111:B4, Cat. No. L2630, Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 vehicle or RPMI 1640 vehicle alone 24 h after M. vaccae ATCC 15483 or BBS vehicle exposure. A fresh stock solution of LPS was prepared in RPMI 1640 to a concentration of 13.25 μg/mL. A total of 10 μL of 13.25 μg/mL LPS stock or RPMI 1640 was pipetted into each well to reach a final concentration of 250 ng/mL. Plates were swirled gently after all wells were treated. This experiment was conducted twice with similar results.

Fig. 1.

Experimental timeline for Experiment 1 investigating the effects of Mycolicibacterium vaccae ATCC 15483 on the immunophenotype of naïve and lipopolysaccharide-challenged murine bone marrow-derived dendritic cells (BMDCs). A total of 1.9 x 10⁶ cells were extracted on DIV 0. On DIV 0, bone marrow-derived cells were seeded at a density of 2 x 10⁵ cells/mL per well in a 24-well plate and allowed to differentiate into BMDCs in the presence of 20 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) until DIV 7. On DIV 7, BMDCs were exposed to 10 μg/mL, 30 μg/mL, 100 μg/mL, or 300 μg/mL M. vaccae ATCC 15483 or sterile borate-buffered saline (BBS) vehicle. Twenty-four h later, on DIV 8, BMDCs were challenged with 250 ng/mL LPS or RPMI 1640 vehicle without removing the prior conditions. Isolation of total RNA was conducted 24 h later on DIV 9. Abbreviations: BMDC, bone marrow-derived dendritic cell; DIV, days in vitro; GM-CSF, granulocyte-macrophage colony stimulating factor; LPS, lipopolysaccharide; RNA, ribonucleic acid.

2.2.3.1. M. vaccae ATCC 15483

This study used a whole-cell, heat-killed preparation of M. vaccae ATCC 15483, i.e., the M. vaccae type strain. M. vaccae ATCC 15483 was purchased from American Type Culture Collection (ATCC) Bonicke and Juhasz (ATCC^® 15483), Manassas, VA, USA). M. vaccae ATCC 15483 was cultured in ATCC^® Medium 1395: Middlebrook 7H9 broth with ADC enrichment at 37 °C, then centrifuged at 3000 × g at 4 °C for ten minutes to pellet the cells; growth media was then removed and cells were weighed and resuspended in sterile BBS to a concentration of 10 mg/ml. Cells were transferred to a sealed sterile glass container and autoclaved at 121 °C for 15 min. Heat-killed M. vaccae ATCC 15483 in sterile BBS stock was stored at 4 °C in sterile glass vials (Lot # LL0119MG0223003).

2.2.3.3. Lipopolysaccharide (LPS)

This study used lipopolysaccharide (LPS, 250 ng/mL; Escherichia coli O111:B4; Cat. No. L2630, Sigma-Aldrich) as an immune challenge, while remaining wells were challenged with RPMI 1640 cell culture media vehicle control condition.

2.2.3.4. Borate-buffered saline (BBS)

This study used sterile BBS as a vehicle control. One-liter sterile BBS was made with 6.19 g of NaCl (Fisher Scientific, Cat. No S642-212), 3.63 g of disodium tetraborate (Na₂B₄O₇·10H₂O) (Sigma Aldrich, Cat. No S9640), 5.25 g boric acid (H₃BO₃) (Fisher Scientific, Cat. No A73-500), 4.7 μL of Tween 80 (polysorbate 80) (AlfaAesar, CAS 9005-65-6), and 800 mL of distilled water. The pH was adjusted to 7.8 using HCl (Fisher Scientific, Cat. No BP153-500). Finally, DI water was added to reach a volume of 1 L.

2.2.4. BMDC preparation

Generation of murine BMDCs was adapted from Lutz et al., (1999). Briefly, mice were euthanized using an overdose of sodium pentobarbital (Fatal Plus^®, Vortech Pharmaceuticals Ltd., Dearborn, MI, USA; 150 mg/kg, i.p.) followed by cervical dislocation. Afterwards, femurs and tibias were dissected and placed in a sterile petri dish (Cat. No. 351029, BD Falcon, Franklin Lakes, NJ, USA) containing 50 mM Dulbecco’s phosphate-buffered saline (Dulbecco’s PBS, Cat. No. D8537, Sigma-Aldrich). Ends of bones were cut above the metaphyses and marrow was flushed into a sterile petri dish using a sterile 10 mL syringe (Cat. No. 309695, BD Falcon) and a sterile 26-gauge needle filled with sterile PBS (Dulbecco’s PBS, Cat. No. D8537, Sigma-Aldrich). Bone marrow cells were transferred to a sterile 50 mL conical tube (Cat. No. 62547205, Sarstedt, Nümbrecht, Germany). Cells were disaggregated using a sterile 1000 μL filtered pipette tip (Cat. No. F123602, Gilson, Middleton, WI, USA). The cell suspension was centrifuged at 500 x g, 10 minutes at room temperature (RT), and the supernatant was discarded. Cells were resuspended in 30 ml of fresh media, which consisted of RPMI 1640 (Cat. No. 10-043-CV, Corning Cellgro, Manassas, VA, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Cat. No. F9423, Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Cat. No. 15140-122, Gibco, Waltham, MA, USA), 50 μM 2-mercaptoethanol (Cat. No. 190242, MP Biomedicals, Santa Ana, CA, USA), and 20 ng/mL GM-CSF (Cat. No. 5191SC, Cell Signaling Technology). Cells were seeded at a density of 2 x 10⁵ cells/mL per well in a 24-well plate (Cat. No. 0030722019, Eppendorf, Hamburg, Germany) in 500 μL of fresh media. Fresh media was added to each well at days in vitro (DIV) 3 (300 μL) and DIV 6 (200 μL), while minimizing the disturbance of the cells. Cells were differentiated over a period of 7 days total before starting experimental procedures.

2.2.5. RNA isolation, and cDNA synthesis

Cell culture plates were centrifuged (Eppendorf, Centrifuge 5810 R, 15 amp version, Cat. No. 022625501; plate rotator S-4-104, Cat. No. 5820755008; plate bucket, Cat. No. 022638930; plate carrier, Cat. No. 5820756004) at 500 x g (1560 r.p.m.) for 5 minutes at 4 °C, then used to harvest RNA by a standard method of guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski & Sacchi, 1987). Briefly, after removing the media, 0.5 ml of TRIzol^® (Cat. No. 15596-026, Invitrogen, Carlsbad, CA, USA; now a brand of the parent company, Thermo Fisher Scientific) was added to each well and plates were frozen at −20 °C until samples were processed. All volumes were adjusted to the initial TRIzol^® amount following the manufacturer’s protocol. Before RNA was precipitated, 10 μg of glycogen (Cat. No. AM9510, Invitrogen) was added to increase RNA precipitation efficiency. RNA reverse transcription was performed using SuperScript II (Cat. No. 18064-014, Invitrogen) following manufacturer’s instructions. RNA was quantified using a NanoDrop spectrophotometer (NanoDrop One C, Thermo Fisher Scientific, Waltham, MA, USA). A volume of 2 μl of RNA sample ranging from 0.5 to 1.5 μg of total RNA was used as template material.

2.2.6. Real-time RT-PCR

Approximately 25 ng of cDNA was used as template material for real-time RT-PCR. Real-time RT-PCR was conducted in duplicate for each well using the CFX96 Touch Real-Time PCR Detection System (Cat. No. 1845097, Bio-Rad, Hercules, CA, USA) and SYBR Green Master Mix (Cat. No. 204145, QuantiTect SYBR Green PCR Kit (1000); QIAGEN, Hilden, Germany). All genes that were analyzed were normalized using the gene Actb (Supplementary Material, Fig. S1), encoding beta actin. Real-time RT-PCR data were represented as a fold increase over the lowest amount of mRNA expressed on the plate for each gene using the 2^−ΔΔCt method. Real-time RT-PCR revealed no effects of either M. vaccae ATCC 15483 or LPS on Actb Cq values (Supplementary Material, Fig. S1), justifying its use as a reference gene.

The Actb primer sequence (Table 1) was designed using the PrimerQuest^™ Tool from the Integrated DNA Technologies (IDT) website (https://www.idtdna.com/pages), designed to span exon/exon boundaries and thus exclude amplification of genomic DNA. The Il10, Il12a, and Il12b primer sequences (Table 1) were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/). Sequence specificity was tested using the Basic Local Alignment Search Tool at NCBI (Altschul et al., 1997). Primers were obtained from IDT (Boulder, CO, USA). Primer specificity was verified by melt curve analyses.

Table 1.

Primers used for real-time RT-PCR listed from 5’ to 3’.

Gene	Forward primer	Reverse primer	Sequence name	Primer source
Actb	TCGTGCGTGACATCAAAG AG	GGATTCCATACCCAAGAA GG	β-actin, cytoskeletal protein (housekeeping gene)	(Desmond et al., 2024)
Il10	GGACTTTAAGGGTTACTTG G	TCACCCAGGGAATTCAAAT G	Interleukin 10 (T-cell growth inhibitory factor)	(Desmond et al., 2024)
Il12a	GCTGATGCAGTCTCTGAAT C	TGAAGCAGGATGCAGAGC TT	Interleukin 12A (encoding IL-12P35)	New
Il12b	AGCACGGCAGCAGAATAA A	AACGCACCTTTCTGGTTAC A	Interleukin 12B (encoding IL-12P40)	New

Abbreviations: Actb, beta actin; Il10, gene encoding interleukin 10; Il12a, gene encoding interleukin 12A; Il12b, gene encoding interleukin 12B.

Ratios of Il10:Il12a and Il10:Il12b mRNA expression were calculated as described previously (Holbrook et al., 2023).

2.3. Experiment 2. Effects of M. sp. strain KGA-10 on the immunophenotype of murine BMDCs

In order to assess the immunoregulatory potential of M. sp. strain KGA-10, we assessed the ability of M. sp. strain KGA-10 to shift the immunophenotype of murine BMDCs toward an anti-inflammatory and immunoregulatory phenotype. Specifically, we evaluated the ability of M. sp. strain KGA-10 to increase the ratios of Il10:Il12a and Il10:Il12b, as these ratios are thought to play an important role in programming DCs toward a regulatory DC (DCreg) phenotype, with potential to induce the differentiation of naïve T cells toward Treg and away from effector T cells, such as Th1 cells, Th2 cells, and Th17 cells, particularly in response to “Old Friends” (Arnold et al., 2012; Rook & Lowry, 2008). We used a concentration of M. sp. strain KGA-10 equivalent to the lowest maximally effective concentration of M. vaccae ATCC 15483, i.e., 100 μg/mL.

2.3.1. Experiment 2. Experimental design: Effects of M. sp. strain KGA-10 and LPS on cytokine mRNA expression in murine BMDCs

Experiment 2 was performed using the same procedures as described above for Experiment 1, with some modifications, described below. The overall experimental design for Experiment 2 is illustrated in Fig. 2. Experiment 2 was a 2 (100 μg/mL M. sp. strain KGA-10 versus sterile BBS vehicle) x 2 (250 ng/mL LPS versus RPMI 1640 vehicle) factorial design. There were eight technical replicates (i.e., eight wells; n = 8) per treatment group. On DIV 0 (5/26/2022), cells were seeded at a density of 2 x 10⁵ cells/mL per well in a 24-well plate and allowed to differentiate in the presence of GM-CSF until DIV 7. On DIV 7, BMDCs were treated with 100 μg/mL M. sp. strain KGA-10 (Lot No.: LL0822EH0822003) or BBS vehicle alone. Stock solutions of M. sp. strain KGA-10 were swirled each time before pipetting into the wells to ensure the suspension was distributed evenly. M. sp. strain KGA-10 treatment consisted of adding 15 μL of a 100 mg/mL stock of M. sp. strain KGA-10 to reach a final concentration of 100 μg/mL in each well, or sterile BBS (vehicle). Twenty-four h after exposure to M. sp. strain KGA-10 or vehicle, i.e., on DIV 8, murine BMDCs, still exposed to prior experimental conditions, were additionally challenged with lipopolysaccharide (LPS; Escherichia coli 0111:B4, Cat. No. L2630, Sigma-Aldrich) in RPMI 1640 vehicle or RPMI 1640 vehicle alone. A fresh stock solution of LPS was prepared in RPMI 1640 to a concentration of 13.25 μg/mL. A total of 10 μL of 13.25 μg/mL LPS stock or RPMI 1640 was pipetted into each well to reach a final concentration of 250 ng/mL. Plates were swirled gently after all wells were treated. This experiment was conducted (N = 1) with eight technical replicates (i.e., eight wells; n = 8) per experimental condition. This experiment was conducted twice with similar results (see Experiment 3).

Fig. 2.

Experimental timeline for Experiment 2 investigating the effects of M. sp. strain KGA-10 on the immunophenotype of naïve and lipopolysaccharide-challenged murine bone marrow-derived dendritic cells (BMDCs). On DIV 0, bone marrow-derived cells were seeded at a concentration of 2 x 10⁵ cells/mL per well in a 24-well plate and allowed to differentiate into BMDCs in the presence of 20 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) until DIV 7. On DIV 7, BMDCs were exposed to 100 μg/mL M. sp. strain KGA-10 or BBS vehicle. Twenty-four h later, on DIV 8, BMDCs were challenged with 250 ng/mL lipopolysaccharide (LPS) or RPMI 1640 vehicle without removing the prior conditions. Isolation of total RNA was conducted 24 h later on DIV 9. Abbreviations: BMDC, bone marrow-derived dendritic cell; DIV, days in vitro; GM-CSF, granulocyte-macrophage colony stimulating factor; LPS, lipopolysaccharide; RNA, ribonucleic acid.

2.3.2. Reagents

2.2.2.1. Mycolicibacterium sp. strain KGA-10

This study used a whole-cell, heat-killed preparation of M. sp. strain KGA-10, which is deposited at the American Type Culture Collection (January 2024; ATCC PTA-127651, a culture deposited January 25, 2024; KGA-10). Briefly, M. sp. strain KGA-10 was originally isolated from soil using techniques outlined by Marshall et al. (2023). Whole-cell, heat-killed M. sp. strain KGA-10 was provided by Mycobacteria Therapeutics Corporation (MTC (Kioga);

Boulder, CO, USA). Purity of the isolate prior to downstream applications was checked by purity streak using Middlebrook 7H10 agar, and 16S rRNA gene amplicon sequencing was used to verify the purity of the final heat-killed preparation of M. sp. strain KGA-10.

2.3.2.2. LPS and BBS

The LPS and BBS used in Experiment 2 were the same as described in Section 2.2.3.3 to Section 2.2.3.4.

2.3.3. BMDC preparation, RNA isolation, cDNA synthesis, and real-time RT-PCR

The procedures for BMDC preparation, RNA isolation, cDNA synthesis, and real-time RT-PCR were the same as described in Section 2.1.4 to Section 2.1.6. Primers for real-time RT-PCR are described in Table 1. There were no differences in Actb mRNA expression among treatment conditions (see Supplementary Material, Fig. S2), justifying its use as a reference gene.

2.4. Experiment 3. Effects of M. sp. strain KGA-10 on biomarkers of hippocampal neuroinflammation, hippocampal microglial priming, and anxiety-like defensive behavioral responses in the rat IS/JSE model

2.4.1. Animals

Adult male Sprague Dawley rats (60-90 days old; 250-274 g) were obtained from Inotiv, Indianapolis, IN, USA. Animals were pair-housed by treatment group with standard rat chow (Harlan Teklad 2918 irradiated rodent chow, Envigo (now Inotiv, Madison, WI, USA) and water in glass bottles available ad libitum provided by University of Colorado Boulder Office of Animal Resources (OAR). The colony was maintained at 22 °C on a 12 h light/dark cycle (lights on from 7:00 a.m. to 7:00 p.m.). Upon arrival on experiment day –28, rats were pair-housed according to the same treatment group in standard Static Allentown micro isolator filter-topped polysulfone rat cages (cage model: Cat. No. PC10198HT, 25.9 cm w × 47.6 cm l × 20.9 cm h; cage top: Cat. No. MBT1019HT, 910 cm²; Static Allentown, Allentown, NJ, USA). The cages contained an approximately 2.5 cm-deep layer of bedding (Cat. No. 7090; Teklad Sani-Chips; Inotiv). Rats in the same cage were labeled by a Sharpie Permanent Marker with ID numbers on their tails. This study was conducted in a manner consistent with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, Eighth Edition (The National Academies Press, 2011). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Boulder. All possible efforts were made to minimize the number of animals used and their suffering.

2.4.2. Experiment 3. Experimental design: Effects of M. sp. strain KGA-10 and IS on anxiety-like defensive behavioral responses and hippocampal neuroinflammation

This study was a 2 (M. sp. strain KGA-10 versus sterile BBS vehicle control) × 2 (IS versus home cage (HC) control condition) experimental design; N = 32 (n = 8 per group). See Fig. 3 for an experimental timeline. Rats received weekly subcutaneous injections of either M. sp. strain KGA-10 in sterile BBS (0.1 mg/0.1 ml) or sterile BBS vehicle control during the period from 9-11 a.m. on experimental days –21, –14, and –7 (see Section 2.3.3; Fig. 3). On experimental day –1, all rats were tested in the juvenile social exploration (JSE) baseline test (see Section 2.3.5; Fig. 3). On experimental day 0, rats were randomly assigned to IS or a HC control condition. Twenty-four hours after IS exposure, rats were tested in the JSE test again (see Section 2.3.4 and Section 2.3.5; Fig. 3). The rats were each returned to their home cage after the JSE test, and they were euthanized immediately for collection of blood and hippocampal tissues (see section 2.3.6).

Fig. 3.

Experimental timeline for Experiment 3. This diagrammatic illustration depicts the experimental timeline for Mycolicibacterium sp. strain KGA-10 or borate-buffered saline (BBS) vehicle treatment in relation to the baseline juvenile social exploration (JSE) test, inescapable tail-shock stress (IS) exposure, the final JSE behavioral test, and euthanasia. N = 32 (BBS/home cage, n = 8; M. sp. strain KGA-10/home cage, n = 8; BBS/IS, n = 8; M. sp. strain KGA-10/IS, n = 8) adult male Sprague Dawley^® rats. Each rat received three weekly immunizations (s.c.) with either 0.1 mg/0.1 mL of whole-cell, heat-killed M. sp. strain KGA-10 in sterile BBS vehicle or 0.1 ml of sterile BBS vehicle; rats were tested in a baseline JSE test on Day –1, then were subsequently exposed to IS or home cage control (HC) conditions on Day 0. Rats were tested in a final JSE test on Day 1, with tissues collected for analysis immediately afterward. Abbreviations: BBS, borate-buffered saline; HC, home cage control conditions; IS, inescapable tail-shock stress; JSE, juvenile social exploration.

2.4.3. Subcutaneous injection of Mycolicibacterium sp. strain KGA-10 and BBS

Heat-killed M. sp. strain KGA-10 was suspended in sterile BBS vehicle to yield a final concentration of 0.1 mg/0.1 mL, and sterile BBS served as the vehicle control. Animals were restrained and received weekly s.c. injections of M. sp. strain KGA-10 (0.1 ml) or BBS vehicle (0.1 ml) on experimental days −21, −14, and −7.

2.4.4. Inescapable tail-shock stress (IS)

Half of the animals (n = 16) were placed in Plexiglas^® tubes (23.4 cm in length; 7 cm in width) and exposed to 100 1.6 mA, 5 s tail shocks with a variable inter-trial interval (ITI) ranging from 30 to 90 s (average ITI = 60 s). All IS exposures were performed between 9–11 a.m. IS animals were returned to their home cages immediately after IS. The other half of animals (n = 16) remained undisturbed in their home cages (HC).

2.4.5. Juvenile social exploration (JSE) test

Inescapable tail-shock stress exposure produces robust decreases in JSE, which is a widely used and validated measure of anxiety-like defensive behavioral responses (File & Seth, 2003) and is sensitive to the neuroinflammatory effects of stress (Goshen & Yirmiya, 2009). JSE was measured 24 h prior to (baseline) and 24 h after (test) IS or HC control conditions during the period from 9-11 a.m. Each animal was transferred to a novel cage containing 2.5 cm-deep shaved wood bedding (Cat. No. 7090; Teklad Sani-Chips; Inotiv) in a dimly lit room (40 lux). After a 15-min habituation period, a 28-32-day-old juvenile male rat was introduced to the experimental subject’s cage for 5 min. Exploratory behaviors performed by the adult rat (sniffing, pinning, licking, and allo-grooming of the juvenile) were timed by an observer blind to treatment condition. After the test, the juvenile was removed, and the adult rat was returned to its home cage. The adult rat was exposed to a novel juvenile during both the baseline JSE test and the final JSE test. Videos were analyzed by M.V.B., who was blinded to treatment condition, using J-watcher (Version 1.0, Copyright C 2000-2006, developed by Macquarie University and University of California, Los Angeles, https://www.jwatcher.ucla.edu/). The duration of time (s) that test rats spent engaged in exploratory behaviors (sniffing, pinning, licking and allo-grooming of the juvenile) were assessed. For each animal, JSE test data were quantified as a percent of baseline JSE. Scores from one blinded rater (M.V.B.) were used for analysis.

$$\text{\%}JSE=\frac{testJSE(\sec )}{baselineJSE(\sec )}$$

2.4.6. Euthanasia and tissue collection

Rats were euthanized immediately after JSE testing (Experimental day 1 between 9-11 a.m.) by receiving an intraperitoneal injection containing a lethal dose of 150 mg/kg, 0.8-1 ml of sodium pentobarbital. Rats were transcardially perfused with 0.9% ice-cold saline to remove leukocytes from the brain. The brain was dissected bilaterally to collect the left and right sides of the hippocampus, which were placed in different tubes. Hippocampus samples were flash frozen in liquid nitrogen and stored at −80 °C for future analysis.

2.4.7. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for assessment of hippocampal mRNA expression

Total RNA was isolated from the left hippocampus utilizing a standard method of guanidinium thiocyanate-phenol:chloroform extraction (Chomczynski & Sacchi, 1987). For detailed descriptions of RNA isolation, cDNA synthesis and PCR amplification protocols to assess hippocampal mRNA expression, refer to our previous publication (Frank et al., 2007). The hippocampus was dissected as described previously (Frank et al., 2018). Briefly, the brain was rapidly extracted, placed on ice and the hippocampus was dissected bilaterally with each hemisphere designated for either mRNA analysis (left hippocampus) or protein analysis (right hippocampus). In the current study, both the left and right hippocampus were used for mRNA analysis, due to low mRNA yield in the left hippocampus. The entire hippocampus was collected given our prior findings of robust stress-induced priming effects in this region (Frank et al., 2018; Frank et al., 2015). The choroid plexus was removed from the hippocampus prior to tissue processing and the hippocampus was flash frozen in liquid nitrogen and stored at −80 °C. Previous studies have shown a main effect of M. vaccae NCTC 11659 on hippocampal Il4 mRNA and IL-4 protein in the dorsal, intermediate, and ventral subdivisions of the hippocampus, without any regional differences. Therefore, here we studied the entire hippocampus, without analyzing the dorsal, intermediate, and ventral subdivisions separately, in order to maximize mRNA yield. In the current study, both the left and right hippocampus were used for mRNA analysis, due to low mRNA yield in the left hippocampus. Total RNA was reverse transcribed into cDNA using the SuperScript II First Strand Synthesis System for RT-PCR (SuperScript^™ II Reverse Transcriptase, Invitrogen, Cat. No: 18064014, Waltham, MA, USA). cDNA sequences were obtained from Genbank at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov). Primer sequences were designed using the Operon Oligo Analysis Tool (http://www.operon.com/technical/toolkit.aspx) or the Integrated DNA Technologies (IDT) PrimerQuest tool (https://www.idtdna.com/pages/tools/primerquest) and tested for sequence specificity using the Basic Local Alignment Search Tool at NCBI (Altschul et al., 1997). Primers were obtained from Invitrogen (Actb, Hmgb1, Il4, Mrc1, Nfkbia, Nlrp3; a brand under the Thermo Fisher Scientific corporation), and IDT (Cd200, Cd200r1, Il12a). Primer specificity was verified by melt curve analyses. All primers were designed to span exon/exon boundaries and thus exclude amplification of genomic DNA (see Table 2 for primer description and sequences). PCR amplification of cDNA was performed using the QuantiFast SYBR Green PCR Kit (2000) (Cat. No. 204056, Qiagen, Hilden, Germany). Formation of PCR product was monitored in real time using the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Real-time RT-PCR was performed to analyze the relative expression of Cd200, Cd200r1, Hmgb1, Il4, Il12a, Mrc1, Nfkbia, and Nlrp3 mRNA (Table 2). Relative expression of mRNA was determined by taking the expression ratio of the gene of interest to Actb, which encodes β-actin, and calculated using the 2^−ΔΔCt method (Livak & Schmittgen, 2001). There were no differences in Actb mRNA expression among treatment conditions, justifying its use as a reference gene (see Supplementary Material Fig. S3).

Table 2.

Primers used for real-time RT-PCR listed from 5’ to 3’.

Gene	Forward primer	Reverse primer	Function	Primer source
Actb	TCGTGCGTGACATCAAA GAG	GGATTCCATACCCAAG AAGG	Cytoskeletal protein (housekeeping gene)	(Frank et al., 2018; Loupy et al., 2021)
Cd200	CTCTCTATGTACAGCCC ATAG	GGGAGTGACTCTCAGT ACTAT	Neuronal antigen that binds CD200R1 to inhibit microglial function	(Frank et al., 2018; Loupy et al., 2021)
Cd200r1	TAGAGGGGGTGACCAA TTAT	TACATTTTCTGCAGCCA CTG	Cognate receptor for CD200 that inhibits microglial function	(Frank et al., 2018; Loupy et al., 2021)
Hmgb1	GAGGTGGAAGACCATG TCTG	AAGAAGAAGGCCGAA GGAGG	Damage associated molecular pattern (DAMP); biomarker of microglial priming	New
Il4	GAACTCACTGAGAAGC TGCA	GAAGTGCAGGACTGCA AGTA	Anti-inflammatory cytokine in the CNS	(Frank et al., 2018; Loupy et al., 2021)
Il12a	TGTCAATCACGCTACCT CCTC	CTTGGCAGGTCCAGAG ACTC	Proinflammatory cytokine	New
Mrc1	AATGGGTGCCTCCCTGG TTT	AGGGTCACCCGTTTTCC AGT	Receptor for mannose that is induced by IL-4	(Frank et al., 2018; Loupy et al., 2021)
Nfkbia	CACCAACTACAACGGC CACA	GCTCCTGAGCGTTGAC ATCA	Induced by NFkB to inhibit NFkB function; biomarker of neuroinflamm ation and microglial priming	(Frank et al., 2018; Loupy et al., 2021)
Nlrp3	AGAAGCTGGGGTTGGT GAATT	GTTGTCTAACTCCAGC ATCTG	Inflammasome component mediating caspase-1/IL-1β activation; biomarker of microglial priming	(Frank et al., 2018)

Abbreviations: Actb, gene encoding beta actin; Cd200, gene encoding CD200; Cd200r1, gene encoding CD200 receptor 1; Hmgb1, gene encoding high-mobility group box 1; Il, interleukin; Mrc1, gene encoding mannose receptor C-type 1; Nfkbia, gene encoding nuclear factor kappa light chain enhancer of activated B cells inhibitor alpha; Nlrp3, gene encoding NLR family pyrin domain containing 3.

2.4.8. Statistical analysis and data presentation

2.4.9. Statistical analysis

Statistical analyses of real-time RT-PCR data were conducted as described below. All statistical analyses were performed in IBM SPSS Statistics (ver. 31.0.0.0). All graphs were made using GraphPad Prism (ver. 10.6.0).

2.4.9.1. Experiment 1: Real time RT-PCR analysis of M. vaccae ATCC 15483 BMDC data

Relative gene expression was calculated using the 2^−ΔΔCt method, followed by a log₁₀ transformation. Log10 transformed data were analyzed for outliers using a two-tailed (α = 0.05) Grubbs’ test for single outliers (Grubbs, 1950, 1969). Extreme outliers were removed prior to graphical representation of the data and statistical analysis (see Supplementary File 1). Relative expression data were analyzed using a multifactor ANOVA followed by Bonferroni’s test for planned post hoc pairwise comparisons (two-tailed alpha level = 0.05). Specifically, in the presence of a main effect of M. vaccae ATCC 15483, planned pairwise comparisons between the BBS control condition and different concentrations of M. vaccae ATCC 15483 conditions were conducted; in the presence of a main effect of LPS challenge, planned pairwise comparisons between RPMI 1640 vehicle and LPS challenge within the same M. vaccae ATCC 15483 condition were conducted.

2.4.9.2. Experiment 2: Real-time RT-PCR analysis of M. sp. strain KGA-10 BMDC data

Relative gene expression was calculated using the 2^−ΔΔCt method, followed by log₁₀ transformation. Log10 transformed data were analyzed for outliers using a two-tailed (α = 0.05) Grubbs’ test for single outliers (Grubbs, 1950, 1969). Extreme outliers were removed prior to graphical representation of the data and statistical analysis (see Supplementary File 1). Relative expression data were analyzed using multifactor ANOVA followed by planned post hoc pairwise comparisons using Fisher’s LSD test (two-tailed alpha level = 0.05).

2.4.9.3. Experiment 3: Effects of M. sp. strain KGA-10 on systemic inflammation, neuroinflammation, and anxiety-like defensive behavioral responses

Extreme outliers were detected using a two-tailed (α = 0.05) Grubbs’ test for single outliers (Grubbs, 1950, 1969) and removed prior to graphical representation of the data and statistical analysis (see Supplementary File 1). If Actb was identified as an outlier, its corresponding genes were excluded from the dataset as well. Shapiro-Wilk’s test was used for testing normality. Analysis was done using multifactor ANOVA to determine the effects of M. sp. strain KGA-10, IS, and M. sp. strain KGA-10 × IS interactions on JSE and hippocampal mRNA expression. If a main effect or interaction was found, planned post hoc pairwise comparisons were made using Fisher’s LSD test.

3. Results

3.1. Experiment 1: Effects of M. vaccae ATCC 15483 on the immunophenotype of murine BMDCs

Previous studies suggest that microbial “Old Friends”, including M. vaccae ATCC 15483, provide protection against inappropriate inflammation by inducing regulatory DCs (DCregs), which in turn promote the differentiation of naïve T cells toward a Treg phenotype (Rook & Lowry, 2008). Induction of a DCreg phenotype by “Old Friends” is characterized by an increase in IL-10 relative to IL-12 (Arnold et al., 2012). Therefore, here we assessed the effects of M. vaccae ATCC 15483 on Il10, Il12a, Il12b, Il10:Il12a, and Il10:Il12b mRNA expression in naïve and LPS-challenged murine BMDCs to evaluate the potential of M. vaccae ATCC 15483 to induce a bias toward a DCreg immunophenotype.

M. vaccae ATCC 15483 induced a dose dependent increase in the relative expression of Il10 mRNA in both naïve and LPS-challenged murine BMDCs (F_{(4, 80)} = 68.84, p < 0.001; Fig. 4A). In the absence of LPS challenge, M. vaccae ATCC 15483 increased relative Il10 mRNA expression at concentrations of 100 μg/mL (p < 0.05) and 300 μg/mL (p < 0.001) (Fig. 4A). In the presence of LPS challenge, M. vaccae ATCC 15483 increased relative expression of Il10 mRNA at concentrations of 10 μg/mL (p < 0.01), 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001), and 300 μg/mL M. vaccae ATCC 15483 (p < 0.001) (Fig. 4A). Within the same BBS or M. vaccae ATCC 15483 conditions, all LPS-challenged murine BMDCs had higher Il10 mRNA expression (p < 0.001; Fig. 4A).

Fig. 4.

Experiment 1. Effects of treatment with different concentrations of M. vaccae ATCC 15483 (10 μg/mL, 30 μg/mL, 100 μg/mL, or 300 μg/mL) relative to a sterile borate-buffered saline (BBS) vehicle condition on A) Il10, B) Il12a, C) Il12b, D) Il10:Il12a ratio, and E) Il10:Il12b ratio mRNA expression in murine bone marrow-derived dendritic cells (BMDCs) challenged, 24 h later, with either RPMI 1640 vehicle control condition or LPS (250 ng/mL), and assessed 24 h following challenge. Il10, Il12a, and Il12b mRNA were measured using real-time reverse-transcription polymerase chain reaction (real-time RT-PCR), with Actb as a reference. For sample sizes, see Supplementary File 1. Bars represent mean + (A-C), ± (D) or – (E) SEM. The black dots overlaid on each bar represent individual values from their corresponding group. *p < 0.05, **p < 0.01, ***p < 0.001, versus BBS vehicle control group under the same LPS condition, Fisher’s LSD test. ###p < 0.001, versus RPMI 1640 vehicle condition under the same M. vaccae ATCC 15483 concentration, Fisher’s LSD test. Abbreviations: BBS, borate-buffered saline; BMDCs, bone marrow-derived dendritic cells; Il10, gene encoding interleukin 10; Il12a, gene encoding interleukin 12A; Il12b, gene encoding interleukin 12B; LPS, lipopolysaccharide; RPMI, Roswell Park Memorial Institute.

Il12a and Il12b encode the IL-12p35 and IL-12p40 subunits of the heterodimeric cytokine, IL-12. M. vaccae ATCC 15483 induced a dose-dependent increase in the relative expression of Il12a mRNA in both naïve and LPS-challenged murine BMDCs (Fig. 4B). Multifactor ANOVA revealed an interaction between M. vaccae ATCC 15483 and LPS (F_{(4, 82)} = 33.15, p < 0.001) as well as main effects of M. vaccae ATCC 15483 (F(_{4, 82}) = 35.85, p < 0.001) and LPS (F(_{1, 82}) = 6297.56, p < 0.001) on relative expression of Il12a mRNA (Fig. 4B). In the absence of LPS challenge, M. vaccae ATCC 15483 increased relative expression of Il12a mRNA at concentrations of 10 μg/mL (p < 0.001), 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001), and 300 μg/mL (p < 0.001) (Fig. 4B). In the presence of LPS challenge, M. vaccae ATCC 15483 increased relative Il12a mRNA expression at concentrations of 10 μg/mL (p < 0.05), 30 μg/mL (p < 0.05), and 100 μg/mL (p < 0.01) (Fig. 4B). Within the same BBS or M. vaccae ATCC 15483 conditions, all LPS-challenged murine BMDCs had higher Il12a mRNA expression (p < 0.001; Fig. 4B).

M. vaccae ATCC 15483 induced a dose-dependent increase in the relative expression of Il12b mRNA in both naïve and LPS-challenged murine BMDCs (Fig. 4C). Multifactor ANOVA revealed an interaction between M. vaccae ATCC 15483 and LPS (F_{(4, 84)} = 33.73, p < 0.001) as well as main effects of M. vaccae ATCC 15483 (F_{(4, 84)} = 102.47, p < 0.001) and LPS (F_{(1, 84)} = 5008.57, p < 0.001) on relative expression of Il12b mRNA. In the absence of LPS challenge, M. vaccae ATCC 15483 increased relative expression of Il12b mRNA at concentrations of 10 μg/mL (p < 0.001), 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001), and 300 μg/mL (p < 0.001; Fig. 4C). In the presence of LPS challenge, M. vaccae ATCC 15483 increased relative Il12b mRNA at concentrations of 100 μg/mL (p < 0.001), and 300 μg/mL (p < 0.001) (Fig. 4C). Within the same BBS or M. vaccae ATCC 15483 concentration, all LPS-challenged murine BMDCs had higher Il12b mRNA expression (p < 0.001; Fig. 4C).

Multifactor ANOVA revealed an interaction between M. vaccae ATCC 15483 and LPS (F_{(4, 85)} = 27.21, p < 0.001) as well as main effects of M. vaccae ATCC 15483 (F_{(4, 85)} = 9.95, p < 0.001) and LPS (F_{(1, 85)} =215.14, p < 0.001) on the Il10:Il12a ratio. The effects of M. vaccae ATCC 15483 on the Il10:Il12a ratio were dependent on whether it was evaluated in the absence or presence of LPS challenge (Fig. 4D). In the absence of LPS challenge, M. vaccae ATCC 15483 decreased the Il10:Il12a ratio at all concentrations studied. Specifically, in the absence of LPS challenge, M. vaccae ATCC 15483 decreased the Il10:Il12a ratio at concentrations of 10 μg/mL (p < 0.001), 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001) and 300 μg/mL (p < 0.001) (Fig. 4D). However, in the presence of LPS challenge, M. vaccae ATCC 15483 increased the Il10:Il12a ratio, suggesting an M. vaccae ATCC 15483-induced a shift of BMDCs toward an immunoregulatory phenotype. In the presence of LPS challenge, M. vaccae ATCC 15483 increased the Il10:Il12a mRNA expression at concentrations of 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001), and 300 μg/mL (p < 0.001) (Fig. 4D). Within the same BBS or M. vaccae ATCC 15483 concentration, all LPS-challenged murine BMDCs had lower Il10:Il12a mRNA expression ratios (p < 0.001 when treated by BBS, 10 μg/mL, 30 μg/mL, and 100 μg/mL M. vaccae ATCC 15483; Fig. 4D), except for the highest M. vaccae ATCC 15483 concentration (300 μg/mL).

Multifactor ANOVA revealed an interaction between M. vaccae ATCC 15483 and LPS (F_{(4, 84)} = 17.60, p < 0.001) as well as main effects of M. vaccae ATCC 15483 (F(_{4, 84}) = 4.63, p < 0.01) and LPS (F_{(1, 84)} =65.83, p < 0.001) on the Il10:Il12b ratio. In the absence of LPS challenge, M. vaccae ATCC 15483 decreased the Il10:Il12b ratio at all concentrations. Specifically, in the absence of LPS challenge, M. vaccae ATCC 15483 decreased the Il10:Il12b at concentration of 10 μg/mL (p < 0.01), 30 μg/mL (p < 0.05), 100 μg/mL (p < 0.05), and 300 μg/mL (p < 0.001; Fig. 4E). In the presence of LPS challenge, M. vaccae ATCC 15483 increased the Il10:Il12b ratio, suggesting an M. vaccae ATCC 15483-induced a shift of BMDCs toward an immunoregulatory phenotype. In the presence of LPS challenge, M. vaccae ATCC 15483 increased the Il10:Il12b mRNA expression ratio at concentrations of 10 μg/mL (p < 0.05), 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001), and 300 μg/mL (p < 0.001) (Fig. 4E). In the presence of BBS, LPS decreased the Il10:Il12b mRNA expression ratio (p < 0.01) (Fig. 4E). At M. vaccae ATCC 15483 concentrations of 10 μg/mL (p < 0.05), 30 μg/mL (p < 0.001), 100 μg/mL (p < 0.001), and 300 μg/mL (p < 0.001), LPS challenge increased the Il10:Il12b ratio (Fig. 4E).

3.2. Experiment 2: Effects of M. sp. strain KGA-10 on the immunophenotype of murine BMDCs

In Experiment 2, we assessed the effects of the novel soil-derived strain, M. sp. strain KGA-10, relative to BBS vehicle, on Il10, Il12a, and Il12b mRNA expression, as well as Il10:Il12a and Il10:Il12b ratios in naïve and LPS-challenged BMDCs.

Multifactor ANOVA revealed an interaction between 100 μg/mL M. sp. strain KGA-10 and LPS (F(_{1, 26}) = 16.44, p < 0.001), as well as main effects of M. sp. strain KGA-10 (F(_{1, 26}) = 85.62, p < 0.001), and LPS (F_{(1, 26)} = 497.80, p < 0.001) on Il10 mRNA relative expression. Planned post hoc pairwise comparisons revealed that M. sp. strain KGA-10, relative to BBS vehicle, increased Il10 mRNA relative expression under both RPMI 1640 vehicle control and LPS-challenged conditions (p < 0.001; Fig. 5A). LPS, relative to the RPMI 1640 vehicle condition, increased Il10 mRNA relative expression under both BBS vehicle and M. sp. strain KGA-10 conditions (p < 0.001; Fig. 5A).

Fig. 5.

Experiment 2. Effects of Mycolicibacterium sp. strain KGA-10 (100 μg/mL) relative to a sterile borate-buffered saline (BBS) vehicle control condition on A) Il10, B) Il12a, C) Il12b mRNA expression, D) Il10:Il12a ratio, and E) Il10:Il12b ratio in murine bone marrow-derived dendritic cells (BMDCs) 24 h after lipopolysaccharide (LPS) challenge (250 ng/mL) or the RPMI 1640 control condition. For sample sizes, see Supplementary File 1. Il10, Il12a, and Il12b mRNA were measured using real-time reverse-transcription polymerase chain reaction (real-time RT-PCR), with Actb as a reference. For sample sizes, see Supplementary File 1. Bars represent mean + (A-C), ± (D) or – (E) S.E.M. The black dots overlaid on each bar represent individual values from their corresponding group. ^###p < 0.001, versus BBS vehicle control group under the same LPS condition, Fisher’s LSD test. *p < 0.05, ***p < 0.001, versus RPMI 1640 vehicle condition under the same M. sp. strain KGA-10 concentration, Fisher’s LSD test. Abbreviations: BBS, borate-buffered saline; BMDC, bone marrow-derived dendritic cell; Il10, gene encoding interleukin 10; Il12a, gene encoding interleukin 12A; Il12b, gene encoding interleukin 12B; LPS, lipopolysaccharide; RPMI, Roswell Park Memorial Institute.

Multifactor ANOVA revealed an interaction between 100 μg/mL M. sp. strain KGA-10 and LPS (F_{(1, 26)} = 15.47, p < 0.001) as well as main effects of M. sp. strain KGA-10 (F_{(1, 26)} = 38.50, p < 0.001) and LPS (F(_{1, 26}) = 552.65, p < 0.001) on Il12a mRNA relative expression. Planned post hoc pairwise comparisons revealed that M. sp. strain KGA-10, relative to BBS vehicle, increased Il12a mRNA relative expression (p < 0.001; Fig. 5B) under the RPMI 1640 vehicle condition, but not under LPS-challenged conditions. LPS, relative to the RPMI 1640 vehicle condition, increased Il12a mRNA relative expression under both BBS vehicle and M. sp. strain KGA-10 conditions (p < 0.001; Fig. 5B).

Multifactor ANOVA revealed an interaction between 100 μg/mL M. sp. strain KGA-10 and LPS (F(_{1, 28}) = 12.44, p < 0.01) as well as main effects of 100 μg/mL M. sp. strain KGA-10 (F(_{1, 28}) = 105.76, p < 0.001) and LPS (F_{(1, 28)} = 1187.24, p < 0.001) on Il12b mRNA relative expression. Planned post hoc pairwise comparisons revealed M. sp. strain KGA-10, relative to BBS vehicle, increased Il12b mRNA relative expression under both RPMI 1640 vehicle control and LPS- challenged conditions (p < 0.001; Fig. 5C). LPS, relative to the RPMI 1640 vehicle condition, increased Il12b mRNA relative expression under both BBS vehicle and M. sp. strain KGA-10 conditions (p < 0.001; Fig. 5C).

Ratios of Il10:Il12a and Il10:Il12b were calculated as described previously (Holbrook et al., 2023).

Multifactor ANOVA revealed a main effect of M. sp. strain KGA-10 to increase the Il10:Il12a ratio (F(_{1, 25}) = 9.51, p < 0.01). Planned post hoc pairwise comparisons revealed that M. sp. strain KGA-10 increased the Il10:Il12a ratio under the RPMI 1640 vehicle control condition (p < 0.05; Fig. 5D).

Multifactorial ANOVA revealed a main effect of M. sp. strain KGA-10 to increase the Il10:Il12b ratio (F_{(1, 25)} = 4.29, p < 0.05). Planned post hoc pairwise comparisons revealed that M. sp. strain KGA-10 increased the Il10:Il12b ratio under the RPMI 1640 vehicle control condition (p < 0.01; Fig. 5E).

3.3.1. Effects of s.c. M. sp. strain KGA-10 and IS on anxiety-like defensive behavioral responses in the JSE test

We sought to determine if s.c. treatment with M. sp. strain KGA-10 would mitigate the effects of stress-induced exaggeration of anxiety-like defensive behavioral responses in the IS/JSE paradigm. Consistent with previous findings (Frank et al., 2018; Loupy et al., 2021), analysis using 2 × 2 ANOVA revealed an IS × M. sp. strain KGA-10 interaction (F_{(1, 27)} = 5.54, p < 0.05) as well as main effects of IS (F_(1,27) = 20.47, p < 0.001) and × M. sp. strain KGA-10 (F_{(1, 27)} = 4.53, p < 0.05) on time spent exploring the juvenile as a percent of baseline JSE. Planned post hoc pairwise comparisons further confirmed that IS decreased JSE in the BBS vehicle control condition (p < 0.001, Fig. 6) demonstrating an IS-induced increase in anxiety-like defensive behavioral responses. Among rats exposed to IS, M. sp. strain KGA-10-treated rats spent a greater amount of time exploring the juvenile relative to the BBS vehicle-treated control group (p < 0.01, Fig. 6), consistent with an anxiolytic effect of M. sp. strain KGA-10 among IS-exposed rats. Furthermore, among M. sp. strain KGA-10-treated rats, there was no effect of IS on anxiety-like defensive behavioral responses in the JSE test.

Fig. 6.

Experiment 3. Effects of treatment with Mycolicibacterium sp. strain KGA-10 relative to a sterile borate-buffered saline (BBS) vehicle on juvenile social exploration (JSE) behavior assessed 24 h after inescapable tail-shock stress (IS) or home cage (HC) control conditions. Baseline JSE was measured six days after the final immunization with M. sp. strain KGA-10 or BBS, i.e., on experimental day –1. Rats were exposed to IS or HC control conditions twenty-four hours after their baseline JSE testing, i.e., on experimental day 0. Rats were tested in the JSE paradigm twenty-four hours after IS, i.e., on experimental day 1. Sample sizes: BBS/HC, n = 8; BBS/IS, n = 8; MTC/HC, n = 8; MTC/IS, n = 8. Data are presented as a percent of baseline JSE. Black dots represent individual data points. Bars represent mean + SEM. *p < 0.05; **p < 0.01, planned post hoc pairwise comparisons using Fisher’s LSD test. Abbreviations: BBS, borate-buffered saline; HC, home cage; IS, inescapable tail-shock stress; JSE, juvenile social exploration; MTC, Mycolicibacterium sp. strain KGA-10.

3.3.2. Effect of M. sp. strain KGA-10 and IS on anti-inflammatory hippocampal mRNA expression: Il4 and IL-4-responsive genes Cd200, Cd200r1, and Mrc1

Previous studies have shown that s.c. administration of M. vaccae NCTC 11659 increases hippocampal Il4 mRNA expression, hippocampal IL-4 protein expression, as well as hippocampal expression of IL-4-responsive genes including Cd200r1 and Mrc1 in adult male Sprague Dawley and F344XBN rats (Fonken et al., 2018a; Frank et al., 2018), although these effects have not been observed in all studies (Loupy et al., 2021). There were no predicted effects of s.c. administration of M. sp. strain KGA-10, IS, or an M. sp. strain KGA-10 × IS interaction on hippocampal Il4 mRNA expression or IL-4-responsive genes in the present study (for details, see Supplementary Material results Section S2.4. and Fig. S4).

3.3.3. Effect of M. sp. strain KGA-10 and IS on expression of proinflammatory genes in hippocampus: Il12a

Although previous studies evaluating the effects of rapidly growing mycobacteria on hippocampal neuroinflammation have not assessed the effects on Il12a or Il12b mRNA expression, here, given the effects of M. sp. strain KGA-10 on the Il10:Il12a mRNA expression in murine BMDCs, we opted to assess hippocampal Il12a mRNA expression in this study. Il12a mRNA encodes IL-12A, the p35 subunit of the proinflammatory cytokine IL-12 (Kobayashi et al., 1989; Wolf et al., 1991). Multifactor ANOVA revealed an M. sp. strain KGA-10 x IS interaction (F_{(1, 25)} = 5.432, p < 0.05). Post hoc pairwise comparisons revealed that, among IS-exposed rats, M. sp. strain KGA-10 decreased hippocampal Il12a mRNA expression (p < 0.05; Fig. 7A). Similarly, among M. sp. strain KGA-10-treated rats, IS decreased hippocampal Il12a mRNA expression (p < 0.01; Fig. 7A).

Fig. 7.

Experiment 3. Effects of treatment with Mycolicibacterium sp. strain KGA-10 (0.1 mg/0.1 mL) or sterile borate-buffered saline (BBS) vehicle on hippocampal expression of genes involved in neuroinflammation and microglial priming, A) Il12a, Hmgb1, B) Nfκbia, and (C) Nlrp3 hippocampal mRNA expression, measured 24 h after inescapable tail-shock stress (IS) or home cage (HC) control conditions. For sample sizes, see Supplementary File 1. All mRNA was measured using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) using the 2^-ΔΔCt method, with Actb as a reference. Black dots represent individual data points. Bars represent mean + SEM. *p < 0.05, **p < 0.01, planned post hoc pairwise comparisons using Fisher’s LSD test. Abbreviation: BBS, borate-buffered saline; HC, home cage; Hmgb1, gene encoding high-mobility group box 1; Il12a, gene encoding IL-12A, the p35 subunit of interleukin 12; IS, inescapable tail-shock stress; KGA-10, Mycolicibacterium sp. strain KGA-10; Nfkbia, nuclear factor kappa light chain enhancer of activated B cells inhibitor alpha; Nlrp3, NLR family pyrin domain containing 3.

3.3.4. Effects of M. sp. strain KGA-10 and IS on expression of hippocampal genes involved in microglial priming: Hmgb1, Nfkbia, Nlrp3

Previous studies suggest that rapidly growing mycobacteria, including M. vaccae NCTC 11659 and M. vaccae ATCC 15483 decrease the expression of genes involved in hippocampal microglial priming, including Hmgb1, Nfkbia, and Nlrp3 in rats (Fonken et al., 2018a; Frank et al., 2018; Noronha et al., 2022), and mice (Desmond et al., 2025), and, indeed, can prevent IS-induced priming of hippocampal microglia (Frank et al., 2018), and can decrease priming of hippocampal microglia in aged rats (Fonken et al., 2018c; Sanchez et al., 2022). To evaluate the effects of M. sp. strain KGA-10 and IS on expression of hippocampal genes involved in microglial priming, here we assessed hippocampal Hmgb1, Nfkbia, and Nlrp3 mRNA expression.

HMGB1 is an alarmin protein released from hippocampal microglia (Frank et al., 2016; Weber et al., 2015). Previous studies have shown that IS increases hippocampal HMGB1 protein, and that this effect is prevented by prior M. vaccae NCTC 11659 administration (Frank et al., 2018; Weber et al., 2015). Multifactor ANOVA revealed an M. sp. strain KGA-10 x IS interaction, as well as a main effect of IS (F_{(1, 25)} = 7.41 p < 0.05) on relative hippocampal Hmgb1 mRNA expression (F(_{1, 25}) = 0.036, p < 0.05). Planned post hoc pairwise comparisons revealed that among M. sp. strain KGA-10-immunized rats, IS decreased relative hippocampal Hmgb1 mRNA expression (p < 0.01; Fig. 7B).

Nfkbia and Nlrp3 are involved in stress-induced microglial priming and their upregulation is considered to represent a microglial priming phenotype (Frank et al., 2018; Weber et al., 2015). Previous studies have shown that s.c. administration of M. vaccae NCTC 11659 or M. vaccae ATCC 15483 decreases hippocampal Nfkbia and Nlrp3 mRNA expression in both rats (Frank et al., 2018) and mice (Desmond et al., 2025), as well as Nfkbia mRNA expression in freshly isolated hippocampal microglia challenged with LPS ex vivo in aged rats (Fonken et al., 2018c). However, the effects of M. sp. strain KGA-10 on Nfkbia and Nlrp3 mRNA expression have not been determined previously. Multifactor ANOVA revealed that the interaction between M. sp. strain KGA-10 and IS approached statistical significance (F(_{1, 27}) = 3.256, p = 0.082). In addition, there was a main effect of IS (F_{(1, 27)} = 9.391, p < 0.05) on hippocampal Nfkbia mRNA expression (Fig. 7C). A main effect of KGA-10 and IS interaction approached statistical significance (F_{(1, 27)} = 3.26, p = 0.082). Planned post hoc comparisons revealed that, among IS exposed rats, M. sp. strain KGA-10 decreased hippocampal Nfkbia mRNA expression (p < 0.01; Fig. 7C). In addition, among M. sp. strain KGA-10-immunized rats, IS decreased Nfkbia mRNA expression (p < 0.05; Fig. 7C).

Multifactor ANOVA revealed an M. sp. strain KGA-10 x IS interaction on relative hippocampal Nlrp3 mRNA expression (F_{(1, 27)} = 5.88, p < 0.05; Fig. 7D). Planned post hoc pairwise comparisons revealed that among IS rats, M. sp. strain KGA-10 decreased hippocampal Nlrp3 mRNA expression (p < 0.05; Fig. 7D). In addition, among M. sp. strain KGA-10-immunized rats, IS decreased Nlrp3 mRNA expression (p < 0.05; Fig. 7D).

3.3.5. Effects of M. sp. strain KGA-10 and IS on the correlation of expression of hippocampal genes involved in microglial priming

As described above, exposure to acute stressors sensitizes the proinflammatory response of microglia to a subsequent immune challenge. Previous studies suggest that HMGB-1 protein is a potential mediator of stress-induced microglial priming and that HMGB-1 does so via the nucleotide-binding domain, leucine-rich repeat, pyrin domain containing protein 3 (NLRP3) inflammasome. Specifically, previous studies have shown that exposure to IS increases HMGB-1 and NLRP3 protein in the hippocampus and increases release of HMGB-1 protein from isolated hippocampal microglia ex vivo. Functional studies using the HMGB-1 antagonist BoxA injected into the cisterna magna before IS suggest that HMGB-1 signaling is necessary for IS-induced sensitization of microglia. Here, we explored potential relationships between hippocampal Hmgb1 mRNA expression with hippocampal Nlrp3 and Nfkbia mRNA expression. Hippocampal Hmgb1 mRNA expression was positively correlated with both hippocampal Nlrp3 (Fig. 8A) and hippocampal Nfkbia (Fig. 8B) mRNA expression.

Fig. 8.

Experiment 3. Graphs illustrating correlations between hippocampal Hmgb1 mRNA expression and A) hippocampal Nlrp3 mRNA expression and B) hippocampal Nfkbia mRNA expression.

4. Discussion

Previous studies suggest that stress resilience effects of microbial “Old Friends”, including soil-derived mycobacteria, are due in part to their ability to induce DCreg, which in turn bias toward differentiation of naïve T cells toward Treg; here we show that a novel strain of soil-derived Mycolicibacterium, M. sp. strain KGA-10, indeed induces a shift toward a DCreg-like immunophenotype in murine BMDCs in vitro and, when administered to adult male rats in vivo prevents IS-induced increases in anxiety-like defensive behavioral responses, in association with reductions in biomarkers of hippocampal neuroinflammation and microglial priming among rats exposed to IS. First, we demonstrated that the rapidly growing Mycolicibacterium, M. vaccae ATCC 15483, induces, in a dose-dependent manner, a shift toward a DCreg-like phenotype in murine BMDCs, characterized by increases in the Il10:Il12a and Il10:Il12b ratios assessed 24 following LPS challenge. M. sp. strain KGA-10 also increased the Il10:Il12a ratio assessed 24 following LPS challenge. Analysis of the effects of M. sp. strain KGA-10 in vivo revealed that s.c. administration of M. sp. strain KGA-10, as observed previously with other rapidly growing mycobacteria, including M. vaccae NCTC 11659 and M. vaccae ATCC 15483, prevented IS-induced increases in anxiety-like defensive behavioral responses, as assessed in the JSE test. This effect was associated with decreases in Il12a mRNA expression under the IS condition, as well as decreases in biomarkers of hippocampal microglial priming, i.e., Nfkbia and Nlrp3, also under the IS condition. While M. sp. strain KGA-10 had no effect on biomarkers of hippocampal microglial priming in home cage control animals, it did decrease Nfkbia and Nlrp3 in animals exposed to IS. This is consistent with studies showing that freshly isolated hippocampal microglia from animals exposed to IS, but not those exposed to home cage control conditions, responded to in vivo administration of M. vaccae NCTC 11659 with decreases in ex vivo LPS-induced Nfkbia mRNA expression (Frank et al., 2018). Consistent with these findings, M. vaccae NCTC 11659 decreases ex vivo LPS-induced Nfkbia mRNA expression in isolated microglia from aged rats (Fonken et al., 2018a). Thus, the ability of both M. vaccae NCTC 11659 and M. sp. strain KGA-10 to decrease biomarkers of hippocampal microglial priming, such as Nfkbia mRNA expression, can be observed under conditions associated with microglial priming, such as following stress exposure or aging. Together, these data suggest that, among rapidly growing mycobacteria, the capacity to shift DCs toward a DCreg-like phenotype is associated with the capacity to decrease markers of hippocampal neuroinflammation and hippocampal microglial priming in association with the capacity to induce stress resilience effects in the IS/JSE model.

First, we demonstrated that the rapidly growing Mycolicibacterium M. vaccae ATCC 15483 induces, in a dose dependent manner, a shift toward a DCreg-like phenotype in murine BMDCs, characterized by increases in the Il10:Il12a and Il10:Il12b ratios assessed 24 following LPS or vehicle challenge. This finding aligns with previous in vivo studies showing that s.c. immunization with rapidly growing mycobacteria, including M. vaccae NCTC 11659 and M. vaccae ATCC 15483 enhances IL-10 expression and suppresses proinflammatory cytokine responses, promoting an anti-inflammatory, stress-protective immune profile (Fonken et al., 2018c; Langgartner et al., 2023; Reber et al., 2016b). The increase in Il10:Il12a and Il10:Il12b ratios observed here suggests that increasing concentrations of M. vaccae ATCC 15483 progressively promotes DCreg differentiate to immunoregulatory state. This is consistent with the “Old Friends” hypothesis that repeated or higher microbial exposures strengthen IL-10-dominant signaling (Rook, 2023; Rook & Lowry, 2008; Rook et al., 2013).

M. sp. strain KGA-10 also has a main effect to increase both the Il10:Il12a ratio and the Il10:Il12b ratio assessed 24 following LPS or vehicle challenge. This suggests that M. sp. strain KGA-10 also promotes a shift toward an anti-inflammatory or DCreg-like phenotype. These findings make M. sp. strain KGA-10 a promising candidate for exploring its immunoregulatory properties, as well as exploring stress resilience effects in vivo. It should also be noted that under the RPMI control condition (i.e., in the absence of subsequent LPS exposure), M. sp. strain KGA-10 increased Il10 mRNA expression (encoding the anti-inflammatory cytokine, IL-10) but also increased the expression of Il12a and 1l12b (encoding subunits of the proinflammatory cytokine IL-12). This is consistent with previous studies showing that the related Mycolicibacterium strain, M. vaccae NCTC 11659, acutely increases local proinflammatory responses in vivo, including Il1b, Il6, and Tnf expression 12-72 h following exposure (Lowry et al., 2007). Other studies suggest that M. vaccae NCTC 11659 has long-lasting “adjuvant-like” effects, increasing hippocampal Il6 mRNA expression eight days following the final M. vaccae NCTC 11659 injection (Loupy et al., 2021), or increasing IL-6 protein secretion and the ratio of IL-6/IL-10 secretion from anti-CD3-stimulated mesenteric lymph node cells up to five weeks following the final M. vaccae NCTC 11659 injection (Reber et al., 2016b).

In the current study, there were no IS-induced increases in expression of biomarkers of microglial priming, including hippocampal Hmgb1, Nfkbia, and Nlrp3 mRNA expression, in BBS vehicle-treated animals. This is consistent with previous studies showing that, 24 h following IS, there is no effect of IS on Nfkbia mRNA expression in freshly isolated hippocampal microglia (Frank et al., 2018). However, freshly isolated hippocampal microglia from rats exposed to IS, relative to home cage controls, respond with a dose dependent exaggeration of LPS-induced increases in Nfkbia mRNA expression assessed 2 h after LPS challenge, consistent with microglial priming (Frank et al., 2018). Importantly, treatment with M. vaccae NCTC 11659 prior to IS prevented the IS-induced exaggeration of LPS-induced increases in Nfkbia mRNA expression (i.e., prevented the IS-induced microglial priming) (Frank et al., 2018). Thus, some effects of Mycolicibacterium treatment and IS may only be evident 24 h following IS exposure when the primed microglia are exposed to a subsequent acute immune challenge, in line with previous studies of microglial priming (Fonken et al., 2018c). This interpretation is consistent with previous studies suggesting that primed microglia do not necessarily secrete high amounts of proinflammatory cytokines, but when triggered by proinflammatory stimuli (e.g., LPS), they become hyper-reactive, secreting large amounts of cytokines, chemokines, and other reactive molecules (Holtman et al., 2015).

Analysis of the effects of M. sp. strain KGA-10 in vivo revealed that s.c. administration of M. sp. strain KGA-10, as observed previously with other rapidly growing mycobacteria, including M. vaccae NCTC 11659 and M. vaccae ATCC 15483, prevented IS-induced increases in anxietylike defensive behavioral responses, as assessed in the JSE test. The prevention of IS-induced anxiety-like defensive behavioral responses by M. sp. strain KGA-10 is consistent with its effect on neuroinflammatory signaling within the hippocampus (downregulation of proinflammatory markers such as Il12a). Comparable to previous studies of M. vaccae NCTC 11659 and M. vaccae ATCC 15483 (Amoroso et al., 2020; Amoroso et al., 2019; Desmond et al., 2025; Foxx et al., 2020; Frank et al., 2018; Loupy et al., 2021; Reber et al., 2016b), M. sp. strain KGA-10 also elicited a “protective” and stress-resilient behavioral phenotype.

Among rats treated with M. sp. strain KGA-10, IS induced a decrease in hippocampal Hmgb1 mRNA expression. Previous studies have confirmed that IS increases HMGB1 and NLRP3 protein in the hippocampus and increases release of HMGB1 from isolated microglia ex vivo (Weber et al., 2015). A potential role for HGMG1 in hippocampal microglial priming following IS exposure is consistent with previous studies demonstrating that intra-cisterna magna administration of an HMGB1 antagonist (BoxA) prior to IS prevented the stress-induced sensitization of microglial proinflammatory responses to a subsequent ex vivo immune challenge (LPS) (Weber et al., 2015). This suggests that HMGB1 is critical for initiating the priming effect.

In addition, intracerebral administration of biologically active disulfide HMGB1 mimicked the effects of IS, such that microglia isolated from HMGB1-treated rats expressed exaggerated NLRP3 and proinflammatory cytokine expression after LPS challenge, while fully reduced, biologically inactive HMGB1 had no effect (Weber et al., 2015). Together with previous studies (Fonken et al., 2018c; Frank et al., 2018; Weber et al., 2015) the present results suggest that the hippocampal innate immune system can respond to IS as if it were cellular damage, thereby releasing the alarmin HMGB1 in the brain to prime microglia by acting on the NLRP3 inflammasome, in preparation for a later immune challenge. As the exaggerated anxiety- and depressive-like behaviors observed when stressed rats are later exposed to a peripheral immune challenge are linked to this enhanced neuroinflammatory response mediated by primed microglia (Fonken et al., 2018b; Weber et al., 2015), M. sp. strain KGA-10-induced changes in Hmgb1, Nfkbia, and Nlrp3 may play a role in its long-term stress resilience effects.

The ability of s.c. treatment with M. sp. strain KGA-10 to ameliorate IS-induced anxiety-like defensive behavioral responses in the IS/JSE model was associated with decreases in biomarkers of hippocampal microglial priming among IS rats, including decreases in Nfkbia and Nlrp3. Decreases in hippocampal expression of Nfkbia and Nlrp3 are perhaps emerging as some of the most reproducible effects of treatment with rapidly growing mycobacteria, including M. vaccae NCTC 11659 and M. vaccae ATCC 15483 in studies of rats and mice (Desmond et al., 2025; Fonken et al., 2018a; Frank et al., 2018; Noronha et al., 2022). These effects of rapidly growing mycobacteria on biomarkers of hippocampal microglial priming are consistent with studies demonstrating that treatment with M. vaccae NCTC 11659 prevents IS-induced priming of hippocampal microglia evaluated ex vivo (Frank et al., 2018) and decreases aging-associated microglial priming (Fonken et al., 2018c; Sanchez et al., 2022).

We evaluated the ability of M. sp. strain KGA-10 to induce a DCreg-like immunophenotype as a cell-based screening assay to evaluate its potential for stress resilience effects in vivo. We selected this approach as the original conceptual framework for the relevance of the hygiene hypothesis or “Old Friends” hypothesis to stress-related psychiatric disorders envisioned a central role for DCreg and immunoregulation in the long-lasting stress resilience properties of mycolicibacteria (Rook & Lowry, 2008). However, future studies will be required to determine if the effects of M. sp. strain KGA-10 to induce a DCreg-like immunophenotype are sufficient to induce stress resilience effects. For example, previous studies by our group have shown that adoptive transfer of Mycolicibacterium obuense-activated murine DCs can induce a T cellspecific interferon gamma (IFNγ) as well as a non-T cell-specific IL-17 response following restimulation of draining lymph node cell preparations obtained 7 days after adoptive transfer of DCs into naïve recipient mice (Crooks et al., 2016). However, it is likely that the effects of M. sp. strain KGA-10 are multi-targeted and that its stress resilience properties may not be fully recapitulated by adoptive transfer of M. sp. strain KGA-10-activated DCs. For example, previous studies using cell-based assays have established that the related Mycolicibacterium strains M. vaccae ATCC 15483 or M. vaccae NCTC 11659 or their molecular constituents can shift multiple immune cell types toward an anti-inflammatory immunophenotype, including freshly isolated murine peritoneal macrophages (Smith et al., 2019), human monocyte-derived macrophages (Holbrook et al., 2023), and murine BV-2 microglia (Desmond et al., 2024) (for review, see (Amoroso et al., 2021; Kleen et al., 2020)). Nevertheless, future studies should evaluate to what extent M. sp. strain KGA-10-activated DCs can recapitulate the effects of s.c. administration of whole-cell, heat-killed preparations of M. sp. strain KGA-10 on the immunophenotype of hippocampal microglia, microglial priming, and stress resilience.

The current study uses a reductionist approach, provides proof-of-concept that it is possible to isolate single strains of mycobacteria from surface soils, screen the mycobacteria for potential immunoregulatory effects in cell-based assays, and validate immunoregulatory and stress resilience effects in vivo. For the current study, selected M. sp. strain KGA-10 for in vivo screening based on its potential for immunoregulatory effects in cell-based assays. The current study identifies M. sp. strain KGA-10 as a candidate for further development as an immunoregulatory intervention that promotes stress resilience.

Subtle differences in the in vitro effects (in BMDCs) and in vivo effects (in the IS/JSE paradigm) of three different strains of Mycolicibacterium (Table 3) suggest opportunities for development of different strains of Mycolicibacterium for different indications using a precision medicine or precision biotic approach.

Table 3.

Comparison of the effects of different Mycolicibacterium strains in cell-based assays and in vivo studies.

Source, cell-based assay, or in vivo paradigm	M. vaccae NCTC 11659	M. vaccae ATCC 15483T	M. sp. strain KGA-10
Source	Soil-derived (Stanford & Paul, 1973)	Cow’s milk (Boenickse & Juhasz, 1964)	Soil-derived (current study)
Cell-based assays
Murine BMDCs; Il10:Il12a ratio	0 ng/mL LPS: ↑ Il10:Il12a ratio (100 μg/mL M. vaccae NCTC 11659) (Lowry et al., unpublished); 250 ng/mL LPS: ↑ Il10:Il12a ratio (100 μg/mL M. vaccae NCTC 11659) (Lowry et al., unpublished);	0 ng/mL LPS: ↓ Il10:Il12a ratio (10, 30, 100, 300 μg/mL M. vaccae ATCC 15483T) (current study); 250 ng/mL LPS: ↑ Il10:Il12a ratio (30, 100, 300 μg/mL M. vaccae ATCC 15483T) (current study)	0 ng/mL LPS: ↑ Il10:Il12a ratio (100 μg/mL M. sp. strain KGA-10) (current study); 250 ng/mL LPS: no effect on Il10:Il12a ratio (100 μg/mL M. sp. strain KGA-10); (current study)
Murine BMDCs; Il10:Il12b ratio	0 ng/mL LPS: ↑ Il10:Il12b ratio (100 μg/mL M. vaccae NCTC 11659) (Lowry et al., unpublished); 250 ng/mL LPS: ↑ Il10:Il12b ratio (100 μg/mL M. vaccae NCTC 11659) (Lowry et al., unpublished)	0 ng/mL LPS: ↓ Il10:Il12b ratio (10, 30, 100, 300 μg/mL M. vaccae ATCC 15483T) (current study); 250 ng/mL LPS: ↑ Il10:Il12b ratio (10, 30, 100, 300 μg/mL M. vaccae ATCC 15483T) (current study)	0 ng/mL LPS: ↑ Il10:Il12b ratio (100 μg/mL M. sp. strain KGA-10) (current study); 250 ng/mL LPS: no effect on Il10:Il12b ratio (100 μg/mL M. sp. strain KGA-10) (current study)
In vivo paradigms (s.c. administration)
IS/JSE paradigm; JSE behavior	Prevents IS-induced increases in anxiety-like behavior in the IS/JSE model (3 x 0.1 mg) (Frank et al., 2018; Loupy et al., 2021)	Prevents IS-induced increases in anxiety-like behavior in the IS/JSE model (3 x 0.1 mg) (Loupy et al., 2021)	Prevents IS-induced increases in anxiety-like behavior in the IS/JSE model (3 x 0.1 mg) (current study)
IS/JSE paradigm; biomarkers of hippocampal anti-inflammatory signaling (Il4, IL-4 protein)	Increases biomarkers of hippocampal anti-inflammatory signaling under home cage control conditions (3 x 0.1 mg) (increases Il4, IL-4 protein) (Frank et al., 2018)	No effect on hippocampal Il4 under home cage control or IS conditions (3 x 0.1 mg); (Loupy et al., 2021)	N.A.
IS/JSE paradigm; biomarkers of alternative macrophages/ microglia activation (Cd200, Cd200r1, Mrc1 (Cd206))	Increases biomarkers of hippocampal alternative macrophages/ microglia activation under home cage control conditions (3 x 0.1 mg) (increases Cd200r1, and Mrc1 (Cd206)); prevents IS-induced decreases in Cd200r1 in freshly isolated hippocampal microglia (Frank et al., 2018); prevents IS-induced decreases in hippocampal Cd200r1 (3 x 0.1 mg) (Loupy et al., 2021)	No effect on hippocampal Cd200, Cd200r1, Mrc1 (Cd206) under home cage control or IS conditions (3 x 0.1 mg); (Loupy et al., 2021)	N.A.
Prevents IS-induced biomarkers of hippocampal neuroinflammation (Il6, Il12a)	Prevents IS-induced increases in hippocampal Il6 (3 x 0.1 mg) (Loupy et al., 2021)	Prevents IS-induced increases in hippocampal Il6 (3 x 0.1 mg) (Loupy et al., 2021)	Decreases hippocampal Il12a under IS conditions (3 x 0.1 mg); (current study)
IS/JSE paradigm; biomarkers of hippocampal microglial priming (Hmgb1, Nfkbia, and Nlrp3 mRNA expression; HMGB1 protein expression)	Decreases hippocampal Nfkbia, Nlrp3 under home cage control conditions (3 x 0.1 mg); prevents IS-induced increases in hippocampal HMGB1 protein expression (Frank et al., 2018)	No effect on hippocampal Nfkbia, under home cage control or IS conditions (3 x 0.1 mg) (Loupy et al., 2021)	Decreases hippocampal Hmgb1, Nfkbia, Nlrp3 under IS conditions (3 x 0.1 mg) (current study)
IS/JSE paradigm; hippocampal microglial priming (LPS-induced Il1b and Nfkbia mRNA expression in freshly isolated hippocampal microglia)	Prevents IS-induced hippocampal microglial priming (3 x 0.1 mg) (based on ex vivo LPS challenge of isolated hippocampal microglia 24 after IS and measurement of Il1b and Nfkbia mRNA expression 2 h after LPS challenge) (Frank et al., 2018)	N.A.	N.A.

Abbreviations: BMDCs, bone marrow-derived dendritic cells; Hmgb1, gene encoding high-mobility group box 1; Il, interleukin; IS inescapable tail shock stress; LPS lipopolysaccharide JSE, juvenile social exploration; Mrc1, gene encoding mannose receptor C-type 1 (CD206); N.A. Not applicable; Nfkbia, gene encoding nuclear factor kappa light chain enhancer of activated B cells inhibitor alpha; Nlrp3, gene encoding NLR family pyrin domain containing 3; s.c. subcutaneous.

4.1. Limitations

There are several limitations that should be considered when interpreting these results. First, the cell-based assays and the in vivo studies were conducted using BMDCs derived from male mice and in vivo assessment of male rats, respectively. As there is evidence that neuroinflammatory priming following acute stress exposure is differentially regulated in males and females (Fonken et al., 2018b), it will be important in future studies to also evaluate the effects of M. sp. strain KGA-10 in females. Also, in vivo assessment of the stress resilience effects of M. sp. strain KGA-10 in vivo were conducted in adults. It will be important to determine in future studies if administration of M. sp. strain KGA-10 can promote stress resilience when administered at different times throughout the lifespan including prenatal, early life, adolescence, and aged adults. It will also be important to determine if M. sp. strain KGA-10 can provide protection through transgenerational or intergenerational effects, as recently described for M. vaccae ATCC 15483.

4.2. Conclusions

Together, these data suggest that M. sp. strain KGA-10, a novel strain of soil-derived rapidly growing Mycolicibacterium, has the capacity to shift murine BMDCs toward a DCreg-like immunophenotype is associated with the capacity to decrease markers of hippocampal neuroinflammation and hippocampal neuroinflammation, in association with stress resilience effects in the IS/JSE model.

Highlights.

• We successfully isolated Mycolicibacterium sp. strain KGA-10 from soil

• M. sp. strain KGA-10 induced a DCreg phenotype in murine BMDCs

• Inescapable tail-shock stress (IS) increased anxiety-like behavioral responses

• M. sp. strain (s.c.) prevented IS-induced anxiety-like behavior in adult male rats

• M. sp. strain KGA-10 (s.c.) decreased biomarkers of hippocampal neuroinflammation

Abbreviations

Actb

gene encoding beta actin

ANOVA

analysis of variance

BBS

borate-buffered saline

BMDC

bone marrow-derived dendritic cell

Cd200

gene encoding CD200

Cd200r1

gene encoding CD200 receptor 1

CNS

central nervous system

Cq

quantification cycle

DIV

days in vitro

ELISA

enzyme-linked immunosorbent assay

GM-CSF

granulocyte-macrophage colony stimulating factor

HC

home cage

Hmgb1

gene encoding high-mobility group box 1

IACUC

Institutional Animal Care and Use Committee

Ido1

gene encoding indoleamine 2,3-dioxygenase 1

IDT

Integrated DNA Technologies

IL-1β

interleukin-1 beta

IL-4

interleukin 4

Il4

gene encoding interleukin 4

Il10

gene encoding interleukin 10

Il12a

gene encoding interleukin 12a (IL-12 subunit P35 of IL-12p70)

Il12b

gene encoding interleukin 12b (IL-12 subunit P40 of IL-12p70)

IS

inescapable tail shock stress

ITI

inter-trial interval

LPS

lipopolysaccharide

LSD

least significant difference

JSE

juvenile social exploration

Mrc1

gene encoding mannose receptor C-type 1 (CD206)

MST

mountain standard time

MTBC

Mycobacterium tuberculosis complex

MTC

Mycobacteria Therapeutics Corporation

NFκB

nuclear factor kappa-light-chain-enhancer of activated B cells

Nfkbia

gene encoding nuclear factor kappa light chain enhancer of activated B cells inhibitor alpha

Nlrp3

gene encoding NLR family pyrin domain containing 3

OAR

Office of Animal Resources

PTSD

posttraumatic stress disorder

Real-time RT-PCR

real-time reverse transcription-polymerase chain reaction

RNA

ribonucleic acid

R/O

reverse osmosis

RPMI

Roswell Park Memorial Institute

RT

room temperature

s.c.

subcutaneous

SEM

standard error of the mean

UTC

Coordinated Universal Time