Bacterial pathogens that reside within host phagocytes face a hostile intracellular environment characterised by reactive oxygen and nitrogen species, nutrient restriction, and progressive acidification of the phagosomal compartment. Among these pathogens, Salmonella enterica serovar Typhimurium has evolved a sophisticated repertoire of virulence factors that enable it both to invade non-phagocytic cells and to survive and replicate inside macrophages. Central to this intracellular lifestyle are two type III secretion systems (T3SS) encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). SPI-1 effectors primarily mediate invasion, whereas SPI-2 effectors remodel the Salmonella-containing vacuole and modulate host cell signalling to create a permissive niche [1,2].Macrophages constitute the principal cellular niche for systemic Salmonella infection. Recognition of bacterial pathogen-associated molecular patterns by Toll-like receptors initiates a cascade of inflammatory signalling that culminates in the production of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and type I interferons. Paradoxically, Salmonella can exploit aspects of this inflammatory response to enhance its own fitness, while simultaneously deploying effectors that attenuate excessive inflammation and promote an anti-inflammatory, M2-like macrophage phenotype [3,4]. Recent studies have highlighted the role of the SPI-2 effector SteE in driving STAT3 phosphorylation and subsequent expression of IL-4 receptor α, thereby skewing macrophages towards a bacteria-permissive state [5,6].Despite substantial progress, several aspects of the reciprocal transcriptional dialogue remain incompletely understood. In particular, the precise temporal order of pro- and anti-inflammatory cytokine induction, the extent of host metabolic reprogramming required for bacterial replication, and the identity of host transcription factors that are selectively targeted by live versus killed bacteria have not been fully resolved. Dual RNA-sequencing approaches that simultaneously capture host and pathogen transcriptomes have begun to illuminate these interactions [7,8], yet integrated molecular and immunological datasets that link gene expression to protein-level cytokine output, surface phenotype and intracellular survival remain relatively scarce.The present study was therefore designed to characterise, in a systematic fashion, the molecular and immunological events that unfold during the first 24 hours of S. Typhimurium infection of murine macrophages. By combining classical microbiological assays with quantitative cytokine profiling, signalling pathway analysis, surface phenotyping and dual transcriptomic approaches, we sought to define the kinetic hierarchy of host responses and to identify previously under-appreciated molecular features of the interaction. An isogenic T3SS mutant lacking the SPI-1 translocon component SipB was included to distinguish invasion- and secretion-dependent effects from those elicited by surface-exposed bacterial ligands alone.

Bacterial strains and culture conditions. Salmonella enterica serovar Typhimurium strain SL1344 and its isogenic ΔsipB mutant were maintained as glycerol stocks at −80 °C. For infection experiments, bacteria were streaked onto Luria–Bertani (LB) agar, and single colonies were inoculated into LB broth and grown overnight at 37 °C with aeration. Overnight cultures were diluted 1:100 into fresh LB and grown to mid-logarithmic phase (optical density at 600 nm ≈ 0.6–0.8). Bacteria were washed twice in phosphate-buffered saline (PBS) and resuspended in antibiotic-free RPMI 1640 medium before infection. Heat-killed preparations were generated by incubation at 70 °C for 30 min and verified by plating.Cell culture and infection. The murine macrophage-like cell line RAW 264.7 was maintained in RPMI 1640 supplemented with 10 % heat-inactivated foetal bovine serum, 2 mM L-glutamine, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin at 37 °C in a humidified 5 % CO2 atmosphere. Primary bone-marrow-derived macrophages (BMDMs) were generated from 6–8-week-old C57BL/6 mice by culturing bone-marrow cells for 7 days in the presence of 20 ng ml−1 recombinant murine macrophage colony-stimulating factor. For infection, cells were seeded in 24-well or 6-well plates and allowed to adhere overnight. Bacteria were added at a multiplicity of infection (MOI) of 10:1 or 50:1 as indicated. After 30 min of synchronised uptake, extracellular bacteria were killed by addition of 100 µg ml−1 gentamicin for 1 h, after which the concentration was reduced to 10 µg ml−1 for the remainder of the experiment. At designated time points, cells were washed and processed for downstream analyses.Gentamicin protection and intracellular survival assay. Infected monolayers were lysed at 1, 4, 8, 12 and 24 h post-infection (hpi) in 0.1 % Triton X-100. Serial dilutions were plated on LB agar and colony-forming units (CFU) enumerated after overnight incubation. Fold replication was calculated relative to the 1 hpi intracellular load.RNA extraction, RT-qPCR and dual RNA-seq. Total RNA was isolated using TRIzol reagent followed by DNase treatment. Complementary DNA was synthesised with random hexamers and SuperScript III reverse transcriptase. Quantitative PCR was performed with SYBR Green chemistry on a QuantStudio 7 system. Relative expression was calculated by the 2−ΔΔCt method using Gapdh (host) or 16S rRNA (bacteria) as reference genes. For dual RNA-seq, rRNA-depleted libraries were prepared from mixed host–pathogen RNA and sequenced on an Illumina NovaSeq platform. Reads were mapped in parallel to the Mus musculus and S. Typhimurium SL1344 genomes. Differential expression analysis was performed with DESeq2; genes with adjusted p-value < 0.05 and |log2 fold-change| > 1 were considered significantly regulated.Cytokine quantification. Culture supernatants were collected at the indicated times, clarified by centrifugation and stored at −80 °C. Concentrations of TNF-α, IL-6, IL-1β, IL-10, IL-12p70 and IFN-γ were determined using a multiplex bead-based immunoassay (Luminex) according to the manufacturer’s instructions. Selected analytes were confirmed by conventional sandwich ELISA.Western blotting. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined by BCA assay. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with antibodies against phospho-p65 (Ser536), total p65, phospho-p38, total p38, phospho-STAT3 (Tyr705), total STAT3, IκBα and β-actin. Band intensities were quantified by densitometry and normalised to loading controls.Flow cytometry. Infected cells were detached, blocked with anti-CD16/32 and stained with fluorochrome-conjugated antibodies against CD80, CD86, MHC class II (I-A/I-E), PD-L1 and F4/80. Viability was assessed with a fixable viability dye. Data were acquired on a BD FACSCanto II cytometer and analysed with FlowJo software.Statistical analysis. Data are presented as mean ± standard deviation of at least three independent biological replicates. Comparisons between groups were performed by two-way analysis of variance followed by Tukey’s multiple-comparison test. A p-value < 0.05 was considered statistically significant.

Intracellular survival depends on a functional type III secretion system

Wild-type S. Typhimurium SL1344 and the isogenic ΔsipB mutant were taken up by RAW 264.7 macrophages at comparable efficiencies (approximately 38 % of the inoculum at MOI 10). Thereafter, intracellular numbers of wild-type bacteria increased steadily, reaching a 45-fold expansion by 24 hpi. In contrast, the ΔsipB mutant exhibited only a modest 3.2-fold increase over the same interval (Table 1). Heat-killed wild-type bacteria were efficiently cleared and never recovered after the gentamicin step. Primary BMDMs displayed a qualitatively similar pattern, although absolute replication rates were lower. These data confirm that a functional T3SS is required for efficient intracellular replication under the conditions employed.

Table 1: Intracellular bacterial loads (log10 CFU per well) at successive time points after infection of RAW 264.7 macrophages (MOI 10).

Data are mean ± SD of three independent experiments. ND, not detected. Statistical comparison performed by two-way ANOVA with Tukey’s post-hoc test.

Unexpected early induction of IL-10 precedes the classical pro-inflammatory cytokine wave

Multiplex cytokine analysis of culture supernatants revealed a temporally ordered secretory programme. Surprisingly, IL-10 protein was already elevated at 4 hpi in wild-type-infected cultures (185 ± 28 pg ml−1), at a time when TNF-α and IL-6 were only beginning to rise (Table 2). By 8–12 hpi the expected pro-inflammatory cytokines reached their maxima, while IL-10 continued to increase modestly. The ΔsipB mutant elicited substantially lower levels of TNF-α and IL-6 and, conversely, higher sustained IL-10 after 12 h. Heat-killed bacteria induced only minimal cytokine release. This early IL-10 surge in the presence of a functional T3SS was unanticipated and suggested that anti-inflammatory signalling is engaged almost immediately upon bacterial entry rather than solely as a late regulatory response.

Table 2 : Cytokine concentrations (pg ml−1) in supernatants of RAW 264.7 macrophages at 4, 8 and 12 h post-infection (MOI 10).

Values are mean ± SD (n = 3). Heat-killed controls produced <50 pg ml−1 of all analytes at every time point. Statistical comparison at 12 hpi by two-way ANOVA.

Host and bacterial transcriptional programmes reveal reciprocal regulation and an unanticipated suppression of Nr4a1

RT-qPCR confirmed robust induction of bacterial SPI-1 and SPI-2 genes inside macrophages. Transcripts of sopB, sipA and sseB increased 18.4-, 12.7- and 9.3-fold, respectively, between input bacteria and the 4–8 h intracellular population. Concurrently, host genes encoding the pro-inflammatory cytokines Tnf, Il1b and Il6 were strongly upregulated, peaking at 4 h (Table 3). Dual RNA-seq extended these observations to a genome-wide scale and uncovered two unexpected features. First, after the initial inflammatory burst, a subset of interferon-stimulated genes (Isg15, Mx1, Ifit1) declined significantly by 12 h in wild-type-infected cells despite continued presence of bacteria, suggesting active dampening. Second, the nuclear receptor Nr4a1 (Nur77) was markedly repressed (log2 fold-change −2.8 at 8 h) exclusively by live wild-type Salmonella; neither the ΔsipB mutant nor heat-killed bacteria produced this effect. Nr4a1 has been implicated in the resolution of inflammation and in the control of macrophage survival; its selective suppression therefore constitutes a previously unreported host target of T3SS-competent Salmonella.

Table 3: Selected host and bacterial transcript fold-changes (relative to uninfected cells or input bacteria) determined by RT-qPCR at 4 and 8 h post-infection.

Fold-changes are mean ± SD relative to uninfected host cells or to input bacterial RNA (n = 3). Nr4a1 repression and the late decline in Isg15 were statistically significant only for wild-type infection (p < 0.01).

Signalling pathway activation and surface phenotype changes

Western blot analysis demonstrated rapid phosphorylation of NF-κB p65 and p38 MAPK within 30–60 min of wild-type infection; peak phospho-p65 levels were 4.8-fold higher than in ΔsipB-infected cells. STAT3 phosphorylation (Tyr705) rose progressively and was maximal at 8–12 h, coinciding with the period of active SPI-2 expression. Flow-cytometric profiling showed early upregulation of the co-stimulatory molecules CD80 and CD86, followed by a marked increase in PD-L1 surface density after 12 h exclusively in wild-type-infected macrophages (mean fluorescence intensity 3.6-fold above uninfected controls). MHC class II expression increased modestly. Collectively these data indicate that T3SS-competent Salmonella drives an initial inflammatory activation that is subsequently tempered by STAT3-dependent and PD-L1-mediated regulatory pathways.

Table 4: Relative densitometric values for selected phosphorylated signalling proteins and surface marker mean fluorescence intensities (MFI) at 8 h post-infection.

Densitometric ratios are normalised to uninfected controls (set to 1.0). MFI values are likewise expressed as fold-change relative to uninfected cells. All wild-type versus ΔsipB comparisons were significant (p < 0.01).

The present study provides an integrated molecular and immunological portrait of the early interaction between S. Typhimurium and murine macrophages. Consistent with earlier reports, a functional type III secretion system was essential for efficient intracellular replication [1,2]. The magnitude of replication observed (approximately 45-fold over 24 h) aligns with values previously documented for SL1344 in RAW 264.7 cells under comparable conditions [9].A notable and previously under-emphasised finding was the early appearance of IL-10 protein at 4 hpi, preceding the peak of classical pro-inflammatory cytokines. Most published kinetic studies have portrayed IL-10 as a late, regulatory mediator that appears after the inflammatory wave has crested [10]. Our data suggest that T3SS-competent Salmonella can trigger an almost immediate anti-inflammatory circuit. Whether this early IL-10 is produced by the infected macrophages themselves or by a rapid paracrine loop remains to be determined; nevertheless, its temporal precedence implies that the pathogen begins to shape the immune microenvironment from the moment of entry.The progressive phosphorylation of STAT3 and the concomitant upregulation of PD-L1 are in agreement with recent work demonstrating that the SPI-2 effector SteE co-opts host GSK3 to phosphorylate STAT3, thereby promoting an M2-like, bacteria-permissive macrophage state [5,6]. Our observation that PD-L1 surface density rises markedly after 12 h extends this paradigm by linking STAT3 activation to a checkpoint molecule known to restrain T-cell responses. The functional consequences of elevated PD-L1 on Salmonella-infected macrophages for subsequent adaptive immunity warrant further investigation.Dual RNA-seq confirmed the expected induction of SPI-2 genes inside macrophages and the reciprocal host inflammatory response. Two transcriptional features stood out as unanticipated. First, several interferon-stimulated genes declined after their initial induction despite ongoing infection, suggesting active bacterial interference with type I interferon signalling. Second, the nuclear receptor Nr4a1 was selectively and strongly repressed only by live, T3SS-competent bacteria. Nr4a1 has been shown to limit inflammatory cytokine production and to regulate macrophage apoptosis [11]; its suppression may therefore constitute a novel virulence strategy that prolongs the lifespan of the infected cell and dampens excessive inflammation. To our knowledge, targeted repression of Nr4a1 by Salmonella has not been reported previously and merits mechanistic follow-up.Comparison with earlier dual RNA-seq studies of Salmonella–host interactions [7,8] reveals both concordance and divergence. The strong induction of SPI-2 and the early host inflammatory signature are conserved features. However, the kinetic detail of IL-10 protein secretion and the specific repression of Nr4a1 emerge more clearly in the present dataset, possibly because of the denser temporal sampling and the parallel protein-level measurements. Metabolic reprogramming towards fatty-acid oxidation, previously linked to SteE and SPI-2 activity [12], was also evident in our transcriptomic data, reinforcing the concept that Salmonella actively rewires host central metabolism to support its intracellular lifestyle. Limitations Several limitations should be acknowledged. First, the study was conducted exclusively in murine macrophages; extrapolation to human cells or to in vivo infection must be made with caution. Second, although dual RNA-seq provided a genome-wide view, the depth of bacterial transcript coverage remains lower than that of the host, potentially limiting detection of low-abundance virulence transcripts. Third, the functional contribution of Nr4a1 repression and of early IL-10 production was not tested by genetic or pharmacological intervention within this work. Finally, single-cell heterogeneity, known to be pronounced during Salmonella infection [13], was averaged in the bulk assays employed here and may obscure subpopulation-specific programmes.

1. Typhimurium orchestrates a temporally structured interaction with macrophages in which an early anti-inflammatory signal (IL-10) precedes the classical pro-inflammatory cytokine wave, STAT3 and PD-L1 are progressively engaged, and selected host regulatory genes such as Nr4a1 are actively repressed. These events depend on a functional type III secretion system and occur in parallel with the activation of bacterial SPI-2 virulence genes. The identification of early IL-10 induction and Nr4a1 suppression as unanticipated features of the interaction expands the known repertoire of host targets manipulated by Salmonella and suggests new points of vulnerability that could be exploited by host-directed therapeutic strategies.