Monocytes were isolated from fresh buffy coats from healthy anonymous donors, provided by the Blood Bank Salzburg. Peripheral blood mononuclear cells (PBMCs) were obtained by gradient density separation with Histopaque-1077 (Sigma-Aldrich, Vienna, Austria). Monocytes were further purified via magnetic labeling using CD14 MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. Monocytes were then cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS; Biowest, Nuaillé, France) and 1% L-glutamine (Fisher Scientific, Schwerten, Germany). Generally, monocytes were rested between 1 and 4 h after isolation prior to being subjected to further treatment.

H. pylori P12 wild type (Hp) was cultured in a bacterial culture Oxoid 3.5 L bucket (Thermo Fischer Scientific, Vienna, Austria) under microaerophilic conditions generated by using an Oxoid CampyGen 3.5 L Sachet (Thermo Fisher Scientific), and at 37°C on GC agar plates containing 10% horse serum (Biowest, Vienna, Austria). For infection, bacterial cells were harvested from culture plates using cotton buds (Paul Boettger, Bodenmais, Germany) and solubilized in 1 mL PBS (Sigma-Aldrich). Optical density of the solution was determined by spectrophotometric measurement (OD600) on a BioPhotometer Plus device (Eppendorf, Vienna, Austria) and the bacterial cell count was calculated against an in-house calibration curve. The density of bacterial cells was then adjusted according to the experimental requirements. Multiplicities of infection (MOI) of 5, 1 and 0.2 were used.

Monocytes were exposed to stimuli on two consecutive days ( Figure 2A ). On day 1, 1×10⁵ monocytes were seeded in 100 µL of cell culture medium (RPMI-1640, 10% heat-inactivated FCS, 1% L-glutamine) in 96-well flat-bottom cell culture plates and rested for 1-4 h at 37°C. Then, 100 µL of cell culture medium containing either E. coli LPS (O55:B5; final concentration of 5 ng/mL), live bacteria (H. pylori P12) or heat-killed H. pylori cells at MOI 5 were added to each well to a final volume of 200 µL, and incubated for 24 h at 37°C and 5% CO₂. On day 2, supernatants were carefully removed from each well and stored, leaving the adherent monocytes unperturbed at the bottom of the well. Immediately after, 200 µL of fresh medium containing 1% Pen/Strep and either 10 ng/mL final concentration of LPS (challenge), H. pylori (MOI5) or medium only were added. The cells were then cultured for a further 24 h, and supernatants collected for analysis. For RNA and protein expression studies, this second incubation period was terminated 2 h post-challenge.

This experimental setup is based on an in vitro model of innate memory, published by Madej et al. (29). Briefly, monocytes were incubated for 7 days between the two treatments ( Figure 5A ). Similar to the short-term innate response model, monocytes were seeded on day 1 at a density of 1×10⁵ cells in RPMI- 1640 medium containing 5% heat-inactivated human serum type AB (Lonza, Basel, Switzerland) and 1% L-glutamine, and primed with H. pylori at different MOIs (0.2, 1 or 5) for 24 h. On day 2, supernatants were carefully removed and stored, and fresh medium containing 1% Pen/Strep was added to the wells. Cells were then cultured for 6 days, with a change of medium at day 4. On day 8, medium was removed and fresh medium was added to the wells, either containing 5 ng/mL LPS (challenge) or not (control). Monocytes where then cultured for an additional 24 h, and on day 9 the supernatants were collected for analysis.

TNF-α and IL-6 ELISAs (Peprotech, London, United Kingdom) were performed on supernatants harvested from experimentally treated human monocytes, according to the manufacturer’s instructions.

Inflammatory mediator production and secretion by monocytes into the supernatant was measured by Multiplex bead-based assay using the Cytokine/Chemokine/Growth Factor 45-Plex Human ProcartaPlex Panel or the Inflammation 20-Plex Human ProcartaPlex Panel (Thermo Fisher Scientific). Briefly, mediator-binding beads were washed once (PBS, 0.05% Tween-20), resuspended in Assay Buffer and distributed in wells of a 96-well V-bottom plate. Aliquots of standards or samples (15 µL) were added, and the plate was incubated on an orbital shaker at 4°C in the dark overnight. The plate was then washed three times with 150 µL/well wash buffer and incubated with 15 µL/well of Detection Antibody solution on an orbital shaker for 30 min at room temperature (RT). The plate was again washed three times and incubated for another 30 min with 20 µL/well of Streptavidin-PE solution. After washing (3x as above), Reading Buffer was added to the wells for analysis. Quantification was performed on a Luminex MagPix instrument (Luminex, Austin, TX, USA), and data were analyzed with ProcartaPlex Analyst software (Thermo Fisher Scientific).

Total cellular RNA was extracted with TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Pure RNA was then reverse transcribed utilizing RevertAid H Minus M-MuLV reverse transcriptase (Thermo Fisher Scientific). Expression levels of genes of interest were determined by quantitative real-time PCR on a Rotor Gene 3000 instrument (Corbett Research, Cambridge, UK), using Luna Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, USA). mRNA expression was normalized to the expression of a housekeeping gene (RPLP0). Relative mRNA expression was calculated as 2^-ΔCt, where the ΔCt value represents the difference between the threshold cycle (Ct) of the gene of interest minus the threshold cycle of the housekeeping gene. Specificity of primers was monitored via analysis of product melting curves. The following primer pairs were used: TNF fwd: 5’- caa gcc tgt agc cca tgt tg -3’, TNF rev: 5’- gag gtt gac ctt ggt ctg gta -3’, IL12A fwd: 5’- tca gca tgt gtc cag cgc gca -3’, IL12A rev: 5’- tct ctt cag aag tgc aag gg -3’, IL6 fwd: 5’- gta cat cct cga cgg cat ctc -3’, IL6 rev: 5’- ggc aag tct cct cat tga atc -3’, IL10 fwd: 5’- agg gca ccc agt ctg aga aca -3’, IL10 rev: 5’- cgg cct tgc tct tgt ttt cac -3’.

Cell surface expression of co-stimulatory molecules on pure monocytes was determined by analysis of median fluorescence intensity on a FACS Canto II flow cytometer (BD Biosciences, San Francisco, CA, USA). Cells were seeded in V-bottom plates after isolation and then cultured with the indicated priming stimulus for 24 h, after which the cells were centrifuged and the supernatants discarded. Thereafter, cells were subjected to the indicated challenge stimulus for 20 h. Cells were then washed in PBS before being stained in 30 µL of staining mix for 30 min in the dark at 4°C. Light exposure was minimized throughout to avoid photobleaching. After staining, the cells were washed and fixed with 4% paraformaldehyde for 15 min at 4°C, then washed twice and resuspended in PBS + 2 mM EDTA for analysis. The following antibody-fluorophore conjugates were used: CD14 PerCP Cy-5.5 (MϕP9, BD Biosciences), Fixable Viability Dye eFluor 506 (eBioscience Thermo Fisher Scientific), CD40-FITC (5C3; eBioscience Thermo Fisher Scientific), CD83-PE-Cy7 (HB15e, BD Biosciences). Data analysis was performed using FlowJo 10 software.

Cell lysates were prepared in NP40 sample buffer, containing 150 mM NaCl, 1% Triton X, 50 mM Tris pH 8.0, 1 mM PMSF (protease inhibitor) and phosphatase inhibitor, diluted 1:1 with 2X Laemmli sample buffer (Bio-Rad, Vienna, Austria) containing 5% beta-mercaptoethanol. Samples were then separated on a 4-12% NuPAGE Bis-Tris gel (Invitrogen, Vienna, Austria) and transferred onto a nitrocellulose membrane (0.45 µm). After blocking of non-specific binding sites with 5% skim milk for 1 h at RT under gentle agitation, the membrane was incubated with the appropriate antibody, prepared in 5% bovine serum albumin diluted in Tris-buffered saline containing 0.1% TWEEN20 (TBS-T) or 5% skim milk in TBS-T, overnight at 4°C under gentle agitation. The membrane was then washed and incubated with the appropriate secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at RT under gentle agitation. After washing, the membrane was incubated with West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific), and detection was performed with a ChemiDoc Imager (Bio-Rad). The following primary and secondary antibodies were used according to the manufacturer’s instructions: phospho-STAT1 (Tyr701) (58D6) rabbit mAb #9167, phospho-STAT3 (Tyr705) (D3A7) XP rabbit mAb #9145, STAT1 (D4Y6Z) rabbit mAb #14995, STAT3 (D3Z2G) rabbit mAb #12640 (all from Cell Signaling Technology, Frankfurt, Germany).

Data are represented as symbols indicating mean ± standard deviation (SD) or as bars indicating mean + SD. Statistical analyses were performed via GraphPad Prism 9 Software (GraphPad Software, San Diego, CA, USA). Differences between multiple stimulation groups were analyzed via two-way ANOVA or matched/repeated measures one-way ANOVA including appropriate post-hoc tests. Sample size is indicated in the figure legends. p values <0.05 were considered significant (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001).

In order to analyze the contribution of innate immune cells to the persistent nature of H. pylori infection, we examined the kinetics of H. pylori-induced secretion of inflammatory mediators in comparison to the prototypical bacterial stimulus LPS derived from Escherichia coli (E. coli). As monocytes are among the first immune cells to infiltrate the inflamed gastric mucosa upon H. pylori infection, we focused our investigations on this cell type. The kinetics of cytokine and chemokine release were monitored for 48 h in LPS-stimulated and H. pylori-infected cells ( Figures 1A, B ). While, in general, similar cytokines and chemokines are released in response to both stimuli ( Figure 1B ), we observed time-dependent differences in the secretion levels. Compared to H. pylori, LPS stimulated a more abundant production of TNF-α and IL-12 in the first 2 h after stimulation. Moreover, while we did not observe a difference at 2 h (when production is very low), IL-10 and IL-6 secretion was markedly increased at 4 and 8 h post LPS stimulation compared to H. pylori infection. Starting at 4 h, but most prominently after 8 h to 48 h of stimulation, H. pylori-induced cytokine secretion was significantly higher than that induced by LPS, with the exception of IL-6, which was secreted at similar amounts ( Figure 1A ). As cytokines are potent inducers of JAK/STAT signaling, we monitored STAT activation in response to H. pylori infection. Again, LPS seems to be more potent than H. pylori in inducing STAT1 and STAT3 phosphorylation at early time points (2 h), while after 24 h, H. pylori-infected cells showed increased STAT1 protein and STAT1 and STAT3 phosphorylation levels compared to cells subjected to LPS stimulation ( Figure 1C ). This set of data indicates that H. pylori induces cytokine and chemokine release in a delayed fashion, with substantially increased secretion levels at later time points compared to LPS stimulation.

It is well established that LPS treatment makes immune cells less responsive to a second LPS stimulus. This can be due to early refractoriness of cells, e.g., ligand-induced receptor down-regulation, or to prolonged tolerance caused by epigenetic and/or metabolic reprogramming (27). This limitation of innate/inflammatory responses induced by bacterial components such as LPS (the aforementioned LPS tolerance) is a key mechanism in maintaining homeostasis and preventing destructive reactivity to repeated challenges against the host. Thus, we aimed to identify how an initial H. pylori infection (“priming”) affects the ability of monocytes to respond to a second H. pylori exposure (“challenge”). Monocytes were activated with LPS (5 ng/mL) or H. pylori (MOI 5) for 24 h then washed and challenged with the same stimulus (filled bars) or left untreated (empty bars) for an additional 24 h ( Figure 2A ). As expected, LPS challenge of unprimed cells (light blue bars, Figure 2B ) resulted in the secretion of all tested mediators, while challenge of LPS-primed cells (dark blue bars, Figure 2B ) resulted in a drastic decrease in the production of all cytokines and chemokines examined ( Figure 2B ). LPS-primed cells challenged with medium only (empty bars, Figure 2B ) still secreted low but measurable amounts of IL-10 and IL-12, suggesting that the cells did not completely return to quiescence. H. pylori challenge of unprimed (light orange, Figure 2C ) cells induced the release of high amounts of all tested mediators ( Figure 2C ). The cytokine release was significantly decreased upon repeated infection with H. pylori (dark orange, Figure 2C ), to the levels observed in unchallenged H. pylori-primed cells (empty bars, Figure 2C ). Notably, IL-10 levels remained high in cells previously infected with H. pylori, but again did not increase with re-infection. These data show that H. pylori infection results in general tolerance to a second H. pylori encounter.

As we observed tolerance to repeated H. pylori exposure, we next aimed to identify whether H. pylori-infected cells can still be activated by a different stimulus, i.e., E. coli LPS, or whether H. pylori infection could induce cross-tolerance to LPS, as observed for other Toll-like receptor (TLR) ligands (30, 31). To compare the responsiveness of H. pylori-primed monocytes to LPS-primed cells, we stimulated monocytes with either H. pylori or LPS for 24 h, washed the cells, and then analyzed cytokine mRNA expression as well as the secretion upon a subsequent LPS challenge. As a challenge control, we also monitored cytokine production by primed monocytes exposed to culture medium only (empty bars, Figure 3 ). In the absence of challenge, the mRNA expression of TNF, IL6, IL10 or IL12B was practically undetectable in both LPS- and H. pylori-primed monocytes (empty bars, Figure 3A ). However, as already observed, cells primed with H. pylori still secreted appreciable amounts of cytokines even after a washing step and in the absence of an LPS challenge (empty bars, Figure 3B ). Strikingly, while repeated LPS treatment made the cells unresponsive ( Figures 3A, B , blue bars), H. pylori-primed cells not only retained responsiveness to an LPS challenge, but they showed significantly enhanced expression and secretion of the cytokines TNF-α, IL-6, IL-10 and IL-12 ( Figures 3A, B , orange bars), compared to unprimed monocytes (black bars, Figures 3A, B ) and unchallenged cells (empty bars, Figures 3A, B ). Similarly, the co-stimulatory molecules CD80 and CD83 were expressed at a significantly higher level in H. pylori-primed monocytes (orange bars) compared to unprimed monocytes (black bars) in response to an LPS challenge ( Figure 3C ). Notably, the reverse sequence of stimuli, i.e., LPS priming followed by a subsequent H. pylori challenge, did not result in increased cytokine secretion compared to cells that were challenged with H. pylori only ( Supplementary Figure 1 ).

To understand whether active infection of monocytes with H. pylori is essential to prime cells for a potentiated secondary response to other bacterial stimuli, we stimulated monocytes with either viable or heat-killed H. pylori and analyzed cytokine secretion after 24 h ( Figure 4A ). Priming with heat-killed bacteria for 24 h resulted in the secretion of TNF-α, IL-6, IL-10 and IL-12 at levels comparable to those induced by LPS, but significantly lower than those induced by viable bacteria (except in the case of IL-6, which was similarly induced by all three stimuli) ( Figure 4A ). We then investigated whether the heat-killed H. pylori was still capable of priming monocytes toward an enhanced response to an LPS challenge. Again, monocytes were subjected to priming with viable or heat-killed bacteria, washed after 24 h and challenged with medium alone (control) or containing LPS. While H. pylori priming again resulted in a significant increase in cytokine secretion upon LPS challenge (orange bars, Figure 4B ), priming with heat-killed bacteria (gray bars, Figure 4B ) did not result in a change of responsiveness to LPS compared to unprimed cells (black bars, Figure 4B ), except in the case of IL-6, in which priming with killed bacteria significantly enhanced the response to LPS. Accordingly, analyses of STAT1 and STAT3 revealed that phosphorylation of STAT1 in particular and to a lesser extent STAT3 was strongly enhanced in H. pylori-primed monocytes upon LPS challenge, while priming of monocytes with heat-killed H. pylori did not induce similar effects ( Figure 4C ). Taken together, our data suggest that monocyte responsiveness to LPS can be significantly enhanced by a 24 h pre-exposure to viable H. pylori, while heat-killed bacteria have little or no effect.

To investigate whether H. pylori can induce innate memory, we set up an in vitro model of innate memory as described previously (29) ( Figure 5A ). Monocytes were infected for 24 h with H. pylori at the indicated MOIs, then washed and rested for 6 days, and eventually challenged with LPS for 24 h. As shown in Figure 5B , infection with H. pylori resulted in a dose-dependent increase of cytokine and chemokine secretion after 24 h. After the resting period, monocyte activation subsided, as shown by the comparable cytokine/chemokine levels between primed cells challenged with medium alone (empty bars, Figure 5C ) and unprimed unchallenged controls. Challenge with LPS induced a tolerance-type memory response, most evident in cells primed with the highest H. pylori MOI and only for inflammatory cytokines (TNF-α, IL-6 and IL-12), not for anti-inflammatory IL-10 or for chemokines.