Endotoxin Preconditioning Blunts STAT1 Signaling in Mouse Spleen Cells

A single injection of bacterial endotoxin can leave mouse immune cells noticeably less responsive to a key inflammatory signal a day later, according to a new open-access study published on 29 September 2026 in the journal Cell Signaling, Inflammation & Disease. Researchers led by Sana Mumtaz Sheikh and Thomas Meyer found that mice pretreated with lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, showed a marked suppression of STAT1 signaling in their splenocytes when those cells were subsequently stimulated with interferon-gamma (IFNγ) in the laboratory.

STAT1, or signal transducer and activator of transcription 1, is a transcription factor central to how immune cells respond to interferons. When IFNγ binds its receptor, Janus kinases JAK1 and JAK2 phosphorylate STAT1 at a specific tyrosine residue, tyrosine 701, in the protein’s carboxyl terminus. Phosphorylated STAT1 molecules then form dimers, travel to the nucleus, and bind gamma-activated sequence (GAS) elements in the promoters of target genes, switching on immune-responsive genes. The new work shows that this well-characterized cascade is selectively dampened in animals that have previously encountered LPS in vivo.

Wild-type C57BL/6N mice and transgenic mice expressing an amino-terminal deletion mutant of STAT1, called STAT1-ΔN, received an intraperitoneal injection of either LPS at 5 milligrams per kilogram of body weight or phosphate-buffered saline as a control. Twenty-four hours later, the animals were euthanized, their spleens harvested, and single-cell suspensions of splenocytes prepared. These isolated cells were then stimulated ex vivo for 30 minutes with recombinant murine IFNγ at 50 nanograms per milliliter, LPS at 1 milligram per milliliter, both agents together, or left untreated.

Western blotting of whole-cell protein extracts revealed the central finding: tyrosine-phosphorylated STAT1 was significantly higher in splenocytes from PBS-treated control mice after IFNγ stimulation than in cells from LPS-preconditioned animals. Notably, the activation state of the closely related transcription factor STAT3, measured as the ratio of phosphorylated to total STAT3, was insensitive to LPS pretreatment, indicating a selective effect on STAT1 rather than a general shutdown of STAT signaling. Levels of the transcription factor NF-κB (p65) remained unchanged across all conditions, while the inhibitory protein IκBα decreased slightly in cells treated with LPS or the LPS/IFNγ combination.

Importantly, the inhibitory effect of LPS pretreatment on IFNγ-driven STAT1 activation was observed only in mice expressing the wild-type STAT1 protein. In animals carrying the STAT1-ΔN mutant, which lacks the first 134 amino-acid residues and behaves as a functional knockout for cooperative gene activation, the reduction in the phosphorylated-to-total STAT1 ratio did not reach statistical significance. This suggests that the LPS-induced tolerance mechanism requires a fully functional STAT1 amino terminus, the domain that normally allows two adjacent STAT1 dimers to cooperate in binding DNA and forming the tetrameric complexes needed for robust IFNγ-induced gene expression.

Flow cytometry extended the biochemical findings to individual immune cell populations. Using fluorescence-activated cell sorting with antibody panels for T-cell, B-cell, and myeloid markers, the researchers quantified mean fluorescence intensity ratios of phosphorylated to total STAT proteins. After ex vivo IFNγ stimulation, phospho-STAT1 levels were significantly reduced in CD4-positive T cells, B cells, myeloid-derived cells, and macrophages from LPS-pretreated mice compared with controls. Only the CD8-positive T cell population showed a similar downward trend that failed to reach statistical significance. Phospho-STAT3 was largely unaffected, with the single exception of B cells, where it was downregulated in the LPS-pretreated group. The distribution of immune cell subsets in the spleen itself did not change significantly 24 hours after LPS injection, with all comparisons showing p-values of at least 0.097.

Transcriptomic profiling by RNA sequencing added a genomic dimension to the picture. From a total of 17,295 genes detected, the researchers identified 190 differentially expressed genes between splenocytes from LPS-pretreated and PBS-treated mice without ex vivo stimulation, and 399 differentially expressed genes after combined LPS/IFNγ stimulation, with 87 genes shared between the two comparisons. The largest contrast, 3,224 differentially expressed genes, appeared between LPS/IFNγ-stimulated cells from sham-treated animals and unstimulated cells from LPS-preconditioned mice. Notably, ex vivo stimulation conditions exerted a stronger influence on transcriptional outputs than the preceding in vivo LPS challenge, and Gene Ontology analysis showed that the enriched biological processes were largely related to immune regulation, antimicrobial defense, and inflammatory responses.

Quantitative PCR validation confirmed that LPS preconditioning significantly reduced the expression of six known STAT1 target genes, Ccl2, Cxcl9, Irf1, Nos2, Socs1, and Socs3, compared with PBS controls under LPS, IFNγ, or combined stimulation conditions. The suppressor of cytokine signaling proteins encoded by Socs1 and Socs3 are established negative feedback regulators of the JAK/STAT pathway, and their induction upon LPS exposure appears to contribute to the blunted STAT1 phosphorylation observed in wild-type cells. Interestingly, in the STAT1-ΔN transgenic line, the inhibitory effect on STAT1 target gene expression was less well preserved for all genes except Socs3, which was induced even more strongly in the mutant animals than in wild-type controls.

The EMSA experiments revealed a striking divergence between the two genotypes. In spleen cells from LPS-injected mice expressing STAT1-ΔN, DNA-binding activity to a radiolabeled M67 probe containing a single GAS element was significantly increased even without ex vivo stimulation, suggesting a sustained state of STAT1-ΔN activation 24 hours after LPS exposure. Slow-migrating bands corresponding to STAT3 homodimers and STAT1/STAT3 heterodimers were also detected in these lysates before any in vitro cytokine stimulation. In contrast, the DNA-binding signal in samples from wild-type mice had already disappeared by the same time point. The authors interpret this as evidence that the amino-terminal truncation prevents the normal downregulation of STAT1 signaling that follows LPS preconditioning.

The study’s authors frame these results within the broader phenomenon of LPS tolerance, a hyporesponsive state that macrophages and peripheral blood mononuclear cells enter after exposure to sub-lethal concentrations of LPS, characterized by a diminished capacity to produce pro-inflammatory mediators. While this phenomenon has been studied predominantly in cultured cells, the authors note that LPS-mediated transcriptional repression at the organismal level is less well characterized, and their mouse model with dysfunctional STAT1 signaling was designed to address this gap. They caution that this was an exploratory pilot study with three biological replicates per condition, each consisting of pooled samples from three individual mice, and that no adjustment for multiple testing was applied. The data suggest that an initial innate immune activation can diminish subsequent cytokine-driven signaling across diverse splenic immune cell subsets, potentially serving as a safeguard against excessive inflammatory responses, and the authors call for future research into the negative feedback mechanisms underlying this inhibitory effect.

Subject of Research: How lipopolysaccharide preconditioning suppresses STAT1 signaling in mouse splenocytes

Article Title: In vivo exposure to lipopolysaccharide inhibits STAT1 signaling

Article References: Sheikh, S. M., Staab, J., Wirths, O., Lühder, F., & Meyer, T. (2026). In vivo exposure to lipopolysaccharide inhibits STAT1 signaling. Cell Signaling, Inflammation & Disease, 1(1), Article 7. https://doi.org/10.1186/s44505-026-00006-w

Image Credits: AI Generated

DOI: 10.1186/s44505-026-00006-w

Keywords: lipopolysaccharide, STAT1, interferon-gamma, LPS tolerance, splenocytes, JAK/STAT pathway, SOCS proteins, flow cytometry, RNA sequencing, innate immunity, inflammation, mouse model