The Silent War Within

How Neutrophils Prime the Gut's Defenses Against Salmonella

Introduction
Organoids
Neutrophils
Experiment
Conclusion

Introduction: The Stealthy Invader and the First Responders

Every year, non-typhoidal Salmonella (NTS) serovars like Salmonella enterica Typhimurium (STM) and Enteritidis (SE) cause over 150 million global infections, triggering brutal gastroenteritis characterized by cramping, diarrhea, and fever ¹ ² . Unlike typhoidal strains that cause systemic typhoid fever, NTS serovars wage war primarily in the gut.

For decades, scientists struggled to study these battles in human-relevant contexts. Mice don't naturally develop human-like Salmonella-induced gastroenteritis, and simple cell lines lack the complexity of living intestine ⁶ . Enter intestinal organoids—miniature, lab-grown gut structures derived from human stem cells.

Salmonella Fast Facts

150M+ annual infections
Primary attack site: Gut epithelium
Key serovars: Typhimurium, Enteritidis

These 3D models contain all major epithelial cell types (enterocytes, goblet cells, Paneth-like cells) arranged in functional villus-like structures surrounding a central lumen ⁶ . When combined with immune cells like neutrophils—the body's rapid-response "first aid" team—they unlock secrets of how our gut detects invaders and mounts defenses.

1. Organoids: The Gut in Miniature

Human intestinal organoids mimic the complex structure of real gut tissue.

Beyond Petri Dishes

Unlike flat cell monolayers, organoids self-organize into crypt-villus units bathed in growth factors (EGF, Wnt, R-spondin) within a gelatinous matrix (Matrigel). This preserves tissue polarity, barrier function, and cellular diversity ⁶ ² .

Infection-Ready

Microinjection techniques deliver pathogens like STM directly into the organoid lumen, mimicking natural infection routes. Infected organoids recapitulate disease hallmarks: mucus hypersecretion, epithelial shedding, and inflammatory signaling ² ⁴ .

Organoids bridge the gap between cell cultures and animal models, providing human-relevant infection data.

2. Neutrophils: More Than Just Bacterial Killers

Neutrophils swarm infection sites within hours. Traditional roles include:

Phagocytosis: Engulfing bacteria.
NETosis: Releasing DNA nets (neutrophil extracellular traps).
Degranulation: Spewing antimicrobial peptides (e.g., lipocalin-2, calprotectin) ¹ ³ .

However, in human intestinal organoids (HIOs), neutrophils reduce intraepithelial but not luminal Salmonella burden, hinting at subtler tactics ¹ .

Serovar-Specific Sabotage: Why Strain Matters

Not all NTS strains trigger identical responses. HIO studies reveal:

STM

Provokes aggressive inflammation and host cell death.

SE

Upregulates metabolism/ROS pathways, causing oxidative stress.

Typhoidal

Actively suppresses inflammation to establish stealthy systemic infection ² .

Scanning EM of a neutrophil (yellow) attacking bacteria (purple).

3. The Landmark Neutrophil-Organoid Experiment

Unraveling Neutrophil's Director Role in Epithelial Defense

Lawrence et al. (2022) used a groundbreaking "PMN-HIO" model to dissect how human neutrophils reshape gut responses to STM ¹ .

Methodology: A Step-by-Step Siege

HIO Setup: Human intestinal organoids derived from pluripotent stem cells.
Infection: STM microinjected into the HIO lumen (10^5 CFU).
Neutrophil Recruitment: Primary human neutrophils added to the culture.
Tracking & Inhibition:
- Neutrophils pre-labeled with CFSE dye for visualization.
- Caspase inhibitors (Casp-1, -3, -4) applied to block cell death pathways.
- IL-1β neutralized with antibodies.

Table 1: Bacterial Burden in STM-Infected HIOs

Condition	Luminal STM (CFU)	Intraepithelial STM (CFU)	Epithelial Shedding
HIO alone	4.2 × 10⁶	1.8 × 10⁵	Low
HIO + neutrophils	3.9 × 10⁶	4.3 × 10⁴	High (TUNEL+ cells)

Results: Neutrophils as Conductors of Cell Extrusion

Neutrophils slashed intraepithelial STM by >75% without reducing luminal loads.
Epithelial shedding surged: Accumulated TUNEL+ dead cells required neutrophil caspase-1 activity.
Caspase-1 inhibition spiked intraepithelial STM, proving its role in bacterial control.
IL-1β was the linchpin: Neutrophil-derived IL-1β alone triggered epithelial extrusion. Blocking IL-1β abolished shedding.

Table 2: Impact of Caspase Inhibition on PMN-HIO Defense

Inhibitor Target	Effect on Epithelial Shedding	Effect on Intraepithelial STM
None (Control)	High	Low (4.3 × 10⁴ CFU)
Caspase-1	Abolished	Increased 4-fold
Caspase-3 or -4	Reduced	No significant change

Analysis: A Protective Inflammatory Trade-off

Neutrophils trade limited direct killing for strategic indirect defense: sacrificing infected epithelial cells via IL-1β-driven extrusion. This "scorched earth" tactic deprives STM of its intracellular niche. The discovery shifts neutrophils from frontline soldiers to directors of epithelial immunity.

The Scientist's Toolkit: Key Research Reagents

Reagent/Model	Function	Example in Use
Human Intestinal Organoids (HIOs)	Recapitulates human gut structure/function; enables luminal microinjection	Modeling STM/SE/typhoidal infection differences ²
CFSE-Labeled Neutrophils	Tracks neutrophil transmigration into organoids	Confirmed neutrophil recruitment to STM-infected lumen ¹
Caspase Inhibitors (e.g., VX-765)	Blocks specific cell death pathways	Identified caspase-1 as critical for extrusion ¹
IL-1β Neutralizing Antibodies	Inhibits IL-1β signaling	Validated IL-1β as the extrusion trigger ¹
Gentamicin Protection Assay	Quantifies intracellular bacteria	Compared STM vs. SE invasion in HIOs ²

Conclusion: Rewriting Inflammation's Narrative

The organoid-neutrophil synergy reveals a paradigm shift: inflammation isn't just collateral damage—it's a precision tool. Neutrophils use IL-1β to "prime" the epithelium, ejecting infected cells via caspase-1 to limit bacterial spread. This explains why NTS gastroenteritis involves violent cramping and shedding: it's a calculated sacrifice ¹ ⁵ .

Future Directions

Therapeutic Screening

Targeting IL-1β or caspases to reduce tissue damage.

Personalized Treatments

Based on serovar-specific responses (STM vs. SE).

Microbiome Integration

Explore how commensals modulate neutrophil-epithelial crosstalk ⁶ .

As organoid technology evolves—adding immune cells, nerves, and flow—we move closer to witnessing, in real time, the silent war waged within our guts. And in this war, neutrophils are not just foot soldiers; they are the generals directing the epithelium's transcriptional defenses.

"The gut is not a passive tube but an active fortress. Neutrophils hold its blueprints." — Insights from the PMN-HIO frontline.

Key Takeaways

Neutrophils direct epithelial defenses via IL-1β
Organoids reveal human-specific infection dynamics
Different Salmonella strains trigger distinct responses
Inflammation can be a precise defensive strategy