Chronic stress promotes myeloid bias in hematopoiesis via the lactobacillus-ila-FoxO1 signaling axis Free

Increasing evidence indicates that chronic stress frequently induces alterations in hematopoiesis and promotes inflammatory responses. However, the precise mechanisms through which gut microbiota and their metabolites regulate these stress-induced hematopoietic abnormalities and immune dysregulation remain unclear.

Chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) mouse models were established and validated through behavioral tests. Flow cytometry assessed HSC differentiation. Metagenomic sequencing, metabolomic profiling, FMT, bacterial culture, and gavage experiments defined the role of gut microbiota. RNA-seq and RT-qPCR identified dysregulated transcripts. ELISA quantified cytokine/protein levels. Confocal microscopy, IHC, and western blotting elucidated the mechanism. All animal experiments followed approved IACUC protocols.

We first established mouse models of CRS or CUMS. We observed that chronic stress significantly reduced the ratio of bone marrow HSPCs and drove a myeloid differentiation bias. This bias was specifically characterized by the expansion of GMPs, increased monocyte production, and decreased neutrophils. Concurrently, stressed mice also exhibited multi-organ pathological changes, including spleen atrophy and shortened intestine. Immunohistochemistry and western blot revealed that stress caused a significant decrease in gut microbiota diversity and an obvious damage in intestinal barrier, manifested by downregulation of ZO-1/ZO-2 expression.

To determine the mediating role of the gut microbiota in the aforementioned hematopoietic and pathological alterations, we transplanted fecal microbiota from stressed mice into normal recipient mice. We found that the recipient mice fully recapitulated the hematopoietic abnormalities observed in the donor mice, as well as pathological features such as spleen atrophy and intestinal shortening. Subsequently, we performed metagenomic sequencing on mouse feces, identifying specifically enriched Lactobacillus strains (Lactobacillus johnsonii and Lactobacillus intestinalis) in stressed mice as key effector bacteria. Then, we administered Lactobacillus johnsonii or Lactobacillus intestinalis to mice, which not only successfully mimicked the stress-induced myeloid differentiation bias but also induced intestinal barrier disruption and depression-like behaviors.

Next, to identify the key metabolites, we conducted untargeted metabolomic profiling on serum from stressed mice. This analysis identified indole-3-lactic acid (ILA) as a significantly altered key metabolite. In vivo functional validation experiments demonstrated that exogenous ILA supplementation promoted monocyte differentiation, suppressed neutrophil production, and induced pathological changes in the spleen and intestine. In vitro experiments showed that co-culture of ILA with sorted c-Kit⁺ HSPCs similarly promoted myeloid differentiation.

To elucidate the mechanism, we performed RNA-seq assays by using HSPCs from the in vivo stress model and from in vitro ILA-treated c-Kit⁺ cells. Intersection analysis revealed significant enrichment of the FoxO signaling pathway in both datasets. Further mechanistic validation confirmed that ILA activates this downstream pathway by driving the nuclear translocation of the transcription factor FoxO1 from the cytoplasm. Importantly, the FoxO1 nuclear translocation inhibitor completely reversed the pro-myeloid differentiation effect of ILA, establishing the core causal regulatory role of the “ILA-FoxO1 axis”.

Simultaneously, ELISA detection revealed that chronic stress upregulated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in the bone marrow. Consistent validation results showed that microbiota transplantation, Lactobacillus colonization, or ILA treatment all successfully recapitulated the bone marrow inflammatory phenotype, forming a self-reinforcing vicious cycle composed of “stress-microbiota-metabolite-inflammation.”

This research provides a comprehensive elucidation of the mechanism by which a gut microbial metabolite mediates stress-induced immune dysregulation through precise regulation of FoxO1 nuclear translocation. It establishes the pivotal role of the “Lactobacillus-ILA-FoxO1” axis in linking chronic stress, myeloid differentiation bias, and inflammation, offering novel directions for understanding related pathological mechanisms and identifying intervention targets.