







































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: arirejay@gmail.com; 
 
Cite as: Islam Ariremako Jimoh, TOBECHI BRENDAN NNANNA, TAIWO MICHEAL OLUMUJI, Paul Onyekachi Ukegbu, 
Michael Sewanu Hungbo, and Kanime Hussaini. 2025. “The Gut–NET–ICB Axis: Microbial Metabolite-Mediated Regulation of 
NETosis and Implications for Cancer Immunotherapy”. Asian Journal of Immunology 8 (1):257–268. 
https://doi.org/10.9734/aji/2025/v8i1177. 

 
 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 257-268, 2025; Article no.AJI.147982 
 

 
 

 

 

The Gut–NET–ICB Axis: Microbial 
Metabolite-Mediated Regulation of 

NETosis and Implications for Cancer 
Immunotherapy 

 
Islam Ariremako Jimoh a*, TOBECHI BRENDAN NNANNA b, 

TAIWO MICHEAL OLUMUJI c, Paul Onyekachi Ukegbu d, 
Michael Sewanu Hungbo e and Kanime Hussaini f 

 
a Department of Microbiology, University of Ilorin, Kwara State, Nigeria. 

b Aston Pharmacy School, College of Life and Health Sciences, Aston University,  
Birmingham B4 7ET, UK. 

c Department of Agricultural Extension and Communication Technology, Federal University of 
Technology Akure, Nigeria. 

d Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University, Elele 
Campus, Elele, Rivers State, Nigeria. 

e Department of Animal Breeding and Genetics, Federal University of Agriculture, Abeokuta, Nigeria. 
f Department of Veterinary Medicine, University of Maiduguri, Nigeria. 

 
Authors’ contributions  

 
This work was carried out in collaboration among all authors. All authors read and approved the final 

manuscript. 
 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2025/v8i1177  
 

Open Peer Review History: 
This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,  

peer review comments, different versions of the manuscript, comments of the editors, etc are available here: 
https://pr.sdiarticle5.com/review-history/147982  

 
 

Received: 06/09/2025 
Published: 20/11/2025 

 

Review Article 

https://doi.org/10.9734/aji/2025/v8i1177
https://pr.sdiarticle5.com/review-history/147982


 
 
 
 

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ABSTRACT 
 

Background: Immune checkpoint blockade (ICB) has transformed oncology, but its efficacy is 
limited by primary and acquired resistance. The gut microbiome is a established determinant of ICB 
response, while neutrophil extracellular traps (NETs) have recently emerged as key mediators of an 
immunosuppressive tumor microenvironment (TME) and ICB resistance. The mechanistic link 
between these two phenomena remains elusive. We hypothesize that gut microbial metabolites 
serve as critical systemic messengers that modulate NET formation (NETosis), thereby influencing 
ICB outcomes. 
Methods & Findings: Through a synthesis of recent literature, we delineate a novel "gut-NET-ICB" 
axis. We provide evidence that specific microbiota-derived metabolites directly regulate NETosis. 
Short-chain fatty acids (SCFAs) like butyrate and polyamines such as spermidine suppress NET 
formation by inhibiting histone deacetylases (HDACs), reactive oxygen species (ROS) production, 
and peptidyl arginine deiminase 4 (PAD4) activity. Conversely, certain secondary bile acids can 
promote NETosis. We propose a model wherein a favorable gut microbiome generates a metabolite 
profile that systemically suppresses pathological NETosis, thereby remodeling the TME to enhance 
CD8+ T-cell infiltration and function, creating a state permissive to ICB. Conversely, dysbiosis 
fosters a pro-NETotic environment that drives resistance. 
Conclusion: The modulation of NETosis by gut microbial metabolites represents a crucial 
mechanism underlying the microbiome's impact on cancer immunotherapy. This mechanistic insight 
positions the gut microbiome and NETosis as complementary therapeutic targets. Strategies to 
promote a NETosis-suppressive metabolite profile through dietary interventions, pre/probiotics, or 
postbiotic supplements or to directly inhibit NETosis (e.g., with PAD4 inhibitors) hold significant 
promise for overcoming ICB resistance and improving patient outcomes. 
 

 

Keywords: Gut microbiome; microbial metabolites; neutrophil extracellular traps (NETs); NETosis; 
immune checkpoint blockade; cancer immunotherapy; tumor microenvironment. 

 

1. INTRODUCTION 
 
The advent of immune checkpoint blockade 
(ICB), which targets regulatory pathways such as 
PD-1/PD-L1 and CTLA-4 to reinvigorate anti-
tumor immunity, represents a paradigm shift in 
oncology (Ribas and Wolchok, 2018). Despite 
producing remarkable and durable responses in 
a subset of patients, ICB therapy is hampered by 
the fact that a significant majority of individuals 
experience primary or acquired resistance 
(Mariniello et al., 2025). This stark variability in 
treatment outcomes has spurred intensive 
research into the factors that govern ICB 
efficacy, leading to the seminal discovery that the 
gut microbiome is a critical determinant of 
therapeutic success. 
 
Compelling evidence from both preclinical 
models and clinical studies has established a 
causal relationship between the composition of 
the gut microbiota and response to ICB. Fecal 
microbiota transplantation (FMT) from ICB 
responders can convert non-responders into 
responders, demonstrating the microbiome's 
potent role in modulating the anti-tumor immune 
response (Routy et al., 2018, Davar et al., 2021). 
Concurrently, the role of innate immune players 

in shaping the tumor microenvironment (TME) 
has gained prominence. Neutrophils, and 
specifically their ability to release neutrophil 
extracellular traps (NETs), have been implicated 
in cancer progression and therapy resistance. 
NETs are web-like structures composed of 
decondensed chromatin decorated with histones 
and cytotoxic granule proteins that are extruded 
by neutrophils in a process called NETosis (Liu 
et al., 2025). While physiologically a host 
defense mechanism, aberrant NET formation in 
the TME promotes metastasis, angiogenesis, 
and, crucially, immunosuppression by physically 
impeding T-cell infiltration and directly 
suppressing T-cell cytotoxic function (Ayodele, 
2025, Teijeira et al., 2020). Recent work has 
directly correlated elevated levels of NETs with 
resistance to ICB in both murine models and 
cancer patients (Yang et al., 2020). 
 
A crucial, yet under-explored, link connecting 
these two fields is the functional output of the gut 
microbiome: its dynamic metabolome. Gut 
bacteria metabolize dietary and host-derived 
substrates into a vast array of small molecules, 
including short-chain fatty acids (SCFAs), 
secondary bile acids, polyamines, and 
tryptophan metabolites that can enter systemic



 
 
 
 

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259 

 

 
 

Fig. 1. The Human GUT (Zhang et al., 2025) 
 
circulation and directly influence the function of 
immune cells, both within and outside the gut 
(Kaltenmeier et al., 2021, Parada Venegas et al., 
2019, Ajibola and Ayodele, 2025, Jia et al., ‘). We 
hypothesize that these microbial metabolites 
serve as systemic rheostats for neutrophil activity 
and NETosis. 
 
This review aims to synthesize emerging 
evidence and provide a comprehensive 
mechanistic framework for the "gut-NET-ICB" 
axis. We will explore how specific gut              
microbial metabolites directly and indirectly 
modulate the molecular pathways of NETosis. 
Furthermore, we will propose an integrated 
model wherein a favorable microbiome,             
through its metabolite profile, suppresses 
pathological NET formation, thereby remodeling 
the TME to be more permissive to T-cell-
mediated killing and enhancing ICB efficacy. 
Elucidating this axis not only deepens our 
fundamental understanding of immunotherapy 

resistance but also unveils novel therapeutic 
strategies to modulate the immune system for 
improved cancer treatment. 
 

2. GUT MICROBIAL METABOLITES: KEY 
MEDIATORS OF HOST IMMUNITY 

 

The gut microbiome exerts a profound influence 
on host physiology not merely through its 
presence but through its immense metabolic 
activity. It functions as a virtual endocrine organ, 
converting dietary components and host-derived 
substances into a diverse array of small 
molecules that serve as key communicators with 
the host's immune system (Ayodele, 2025). 
These metabolites can mediate their effects 
locally within the gastrointestinal tract or, upon 
absorption into the portal circulation,       
systemically throughout the body, thereby 
influencing immune responses in distant sites, 
including the tumor microenvironment (TME) (Jia 
et al., 2018). 



 
 
 
 

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260 

 

Table 1. Key classes of microbiota-derived metabolites and their immunomodulatory mechanisms 
 

Metabolite 
Class 

Key Examples Bacterial Origin / Precursor Primary Immunologic Mechanisms Impact on Immunity 

Short-Chain 
Fatty Acids 
(SCFAs) 

Acetate, Propionate, Butyrate Fermentation of dietary fiber by 
genera like Faecalibacterium, 
Roseburia, Lachnospiraceae 
(Mager et al., 2020) 

Roseburia, Lachnospiraceae (Mager 
et al., 2020). - HDAC inhibition 
(Mager et al., 2020) 
- Signaling via GPCRs (GPR41, 
GPR43, GPR109a) (Ayodele et al., 
2025) 
- Energy source for colonocytes & 
immune cells. 

Generally Anti-inflammatory: Promote 
Treg differentiation, maintain gut 
barrier integrity, modulate neutrophil 
chemotaxis and function. 

Secondary Bile 
Acids 

Deoxycholic Acid (DCA), 
Lithocholic Acid (LCA), 
Ursodeoxycholic Acid (UDCA) 

Bacterial dehydroxylation of 
primary bile acids (cholic acid, 
chenodeoxycholic acid) by 
species like Clostridium cluster 
XIVa (Parada Venegas et al., 
2019). 

- Signaling via nuclear receptor FXR & 
membrane receptor TGR5 (Ma et al., 
2025). 

Context-Dependent: Can be pro-
inflammatory (DCA) or anti-
inflammatory (UDCA). Regulate 
metabolic homeostasis and 
macrophage differentiation. 

Polyamines Spermidine, Spermine, 
Putrescine 

Decarboxylation of amino acids 
(e.g., arginine, ornithine) by 
bacteria like Bifidobacterium and 
Lactobacillus (Zhang et al., 
2025). 

- Regulation of autophagy, translation 
(via hypusination) (Qi et al., 2025) 
- Anti-oxidant effects 
- Modulation of ion channels. 

Immunomodulatory: Support T-cell 
differentiation and function, suppress 
pro-inflammatory cytokine production, 
can inhibit NETosis. 

Tryptophan 
Catabolites 

Indole, Indole-3-aldehyde, 
Indolepropionic Acid (IPA) 

Tryptophan metabolism by 
bacteria (e.g., Lactobacillus spp.) 
expressing tryptophanase 
(Tofalo et al., 2019). 

- Activation of the Aryl Hydrocarbon 
Receptor (AhR) (Puleston et al., 
2019). 

Barrier Fortification & Immune 
Regulation: AhR activation 
strengthens epithelial barriers, 
modulates Th17/Treg balance, and 
suppresses innate immune activation. 

Other Bioactive 
Metabolites 

Inosine, D-lactate, 
Trimethylamine N-oxide (TMAO) 

Purine metabolism (Inosine), 
fermentation (D-lactate), 
choline/carnitine metabolism 
(TMAO). 

- Adenosine A2A receptor signaling 
(Inosine) (Kullberg et al., 2025) 
- Precursor to pro-atherogenic TMAO. 

Variable: Inosine enhances Th1 
differentiation and ICB response; 
TMAO is generally pro-inflammatory 
and linked to cardiovascular disease. 



 
 
 
 

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2.1 Short-Chain Fatty Acids (SCFAs): 
Microbial Fermentation Products 
with Systemic Reach 

 

SCFAs are the most extensively studied 
microbial metabolites. Butyrate is a primary 
energy source for colonocytes, crucially 
maintaining gut barrier function and preventing 
systemic inflammation. Beyond this local role, 
SCFAs function as potent epigenetic regulators 
via HDAC inhibition, leading to increased histone 
acetylation and altered gene expression in 
immune cells (Ajibola and Ayodele, 2025, Mager 
et al., 2020). This mechanism promotes the 
differentiation of regulatory T cells (Tregs), which 
are critical for maintaining immune tolerance. 
SCFA signaling through GPCRs like GPR43 on 
neutrophils can also dampen their inflammatory 
potential and chemotaxis, representing a direct 
pathway for systemic immunomodulation 
relevant to NETosis (Ayodele et al., 2025). 
 

2.2 Secondary Bile Acids (BAs): From 
Digestion to Immunomodulation 

 

Primary bile acids, synthesized in the liver, are 
transformed into secondary bile acids by gut 
bacteria. This process significantly alters their 
signaling properties. Secondary BAs act as 
signaling molecules through the nuclear receptor 
FXR and the G-protein-coupled receptor TGR5, 
regulating not only metabolic homeostasis but 
also immune cell function (Ma et al., 2025). For 
instance, specific secondary BAs can promote 
the differentiation of anti-inflammatory 
macrophages, while others, in excess, can be 
cytotoxic and induce inflammatory stress 
responses. 
 

2.3 Polyamines: Crucial for Cellular and 
Immune Homeostasis 

 

Polyamines are essential for cell proliferation and 
are produced by both mammalian cells and gut 
bacteria. Bacterial-derived polyamines can 
influence host immunity by modulating 
autophagy, a process critical for immune cell 
function and antigen presentation (Qi et al., 
2025). Spermidine, in particular, has been shown 
to have anti-inflammatory and life-span-
extending effects, partly through its ability to 
induce autophagy and suppress oxidative stress. 
 

2.4 Tryptophan Metabolites: AhR Ligands 
and Barrier Regulators 

 

Gut bacteria that metabolize the essential amino 
acid tryptophan into ligands for the AhR play a 

vital role in immune education and barrier 
integrity. AhR activation in intestinal epithelial 
cells and intraepithelial lymphocytes strengthens 
the mucosal barrier and helps maintain a 
balanced immune response, preventing aberrant 
inflammation (Puleston et al., 2019). The 
systemic immunologic impact of these 
metabolites is an area of active investigation. 
 

The gut microbial metabolome constitutes a 
critical interface between the external 
environment and the host immune system. The 
balance and composition of these metabolites 
directly influence the functional state of innate 
and adaptive immune cells, creating a systemic 
milieu that can either favor or suppress anti-
tumor immunity. The subsequent sections will 
delve into how these specific metabolites interact 
with the process of NETosis to shape this 
immunologic landscape (Puleston et al., 2019). 
 

3. MECHANISTIC INTERPLAY: 
MICROBIAL METABOLITES AS 
REGULATORS OF NETOSIS 

 

The central hypothesis of the gut-NET-ICB axis 
posits that gut microbial metabolites systemically 
influence the propensity of neutrophils to 
undergo NETosis, thereby shaping the immune 
landscape of the tumor microenvironment (TME). 
This interaction is not merely correlative; a 
growing body of evidence demonstrates direct 
and indirect mechanistic pathways through which 
these bacterial byproducts modulate the 
molecular machinery of NET formation. The 
balance between NETosis-suppressive and 
NETosis-promoting metabolites can critically 
determine the outcome of anti-tumor immunity 
(Ma et al., 2025). 
 

3.1 Suppressive Metabolites: SCFAs and 
Polyamines as NETosis Brakes 

 

Short-Chain Fatty Acids (SCFAs): Butyrate has 
been demonstrated to robustly inhibit NETosis 
induced by various stimuli, including PMA and 
LPS, in human neutrophils in vitro (Zhang et al., 
2025). The primary mechanism is epigenetic: as 
potent HDAC inhibitors, SCFAs lead to 
hyperacetylation of histones. This increased 
negative charge makes the chromatin less 
accessible to PAD4, thereby preventing the 
histone citrullination that is essential for 
chromatin decondensation. Furthermore, SCFA 
signaling through GPR43 on the neutrophil 
surface directly suppresses phorbol ester-
induced ROS production, dismantling a critical 
trigger for the NETotic cascade (Cai et al., 2025). 



 
 
 
 

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Table 2. Mechanisms of Gut Microbial Metabolites in Regulating NETosis 
 

Metabolite Class Net Effect on 
NETosis 

Direct Molecular Mechanisms Indirect Immunological Mechanisms 

Short-Chain Fatty 
Acids (SCFAs) 

Potently Suppressive - HDAC Inhibition: Hyperacetylation of histones and other proteins 
interferes with PAD4 activity and chromatin decondensation (Zhang et 
al., 2025). 
- GPCR (GPR43) Signaling: Downregulates NADPH oxidase assembly 
and ROS production, a key trigger for suicidal NETosis (Cai et al., 
2025). 
- Inhibition of NF-κB Pathway: Reduces transcription of pro-
inflammatory cytokines that prime neutrophils. 

- Promotes differentiation of anti-inflammatory 
Tregs, reducing the cytokine drive for NETosis. 
- Enhances gut barrier integrity, reducing 
systemic translocation of LPS, a potent 
NETosis inducer. 

Polyamines  Potently Suppressive - Direct Inhibition of PAD4: Competitively binds to the enzyme's active 
site, preventing histone citrullination (Perrone and D'Angelo, 2025). 
- Suppression of ROS: Inhibits the assembly and function of the 
NADPH oxidase complex. 
- Induction of Autophagy: Autophagic flux can clear damaged 
mitochondria (mitophagy), preventing mtROS-induced NETosis (Saha 
and Goswami, 2025). 

- Modulates macrophage polarization towards 
an M2-like, anti-inflammatory phenotype. 
- Supports T-cell function, creating a cytokine 
milieu less conducive to persistent neutrophil 
activation. 

Tryptophan Metabolites 
(AhR Ligands) 

Suppressive - AhR Activation: AhR signaling in neutrophils directly upregulates anti-
apoptotic genes and downregulates pro-inflammatory pathways, 
raising the activation threshold for NETosis (Zhao et al., 2025). 

- AhR activation in dendritic cells and T cells 
promotes immune tolerance and a balanced 
cytokine response. 
- Strengthening of the gut barrier reduces 
systemic inflammatory tone. 

Secondary Bile Acids Context-Dependent 
(Often Promotive in 
Dysbiosis 

- Mitochondrial Stress: High concentrations of DCA act as detergents, 
disrupting mitochondrial membranes and inducing mitochondrial ROS 
(mtROS), which drives NETosis (Zhao et al., 2025). 
- NLRP3 Inflammasome Activation: Can prime and activate the 
inflammasome in immune cells, leading to IL-1β release, a potent 
NETosis trigger. 

- Certain BAs can have anti-inflammatory 
effects. An imbalanced ratio of pro- to anti-
inflammatory BAs creates a systemic 
environment that favors neutrophil activation. 



 
 
 
 

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Polyamines: Spermidine emerges as a 
powerful, direct inhibitor of NETosis. It exerts its 
effect by targeting two key nodes in the process. 
First, it directly inhibits PAD4 enzymatic activity, 
preventing the initiation of chromatin 
decondensation (Perrone and D'Angelo, 2025). 
Second, it suppresses the generation of ROS by 
the NADPH oxidase complex. Additionally, its 
well-established role as an inducer of autophagy 
(Saha and Goswami, 2025, Lu et al., 2024) helps 
maintain cellular homeostasis in neutrophils by 
clearing damaged organelles that could 
otherwise serve as intrinsic inducers of NETosis. 
 

3.2 Inductive Metabolites: The 
Detrimental Role of Dysbiosis 

 
Secondary Bile Acids: In a state of dysbiosis, 
an overabundance of certain secondary bile 
acids like deoxycholic acid (DCA) can have the 
opposite effect. At high concentrations, DCA can 
disrupt mitochondrial membranes in neutrophils, 
leading to the leakage of mitochondrial DNA and 
the generation of mitochondrial ROS (mtROS). 
This mtROS can act as a potent trigger for 
NETosis, independent of or synergistic with the 
NADPH oxidase pathway (Saha and Goswami, 
2025). This highlights that the impact of microbial 
metabolites is not universally beneficial and is 
entirely dependent on the compositional and 
metabolic balance of the gut community. 
 

3.3 Indirect Modulation via the Tumor 
Microenvironment 

 
Beyond direct actions on neutrophils, microbial 
metabolites sculpt the broader TME, creating 
feedback loops that influence NETosis. For 
instance, SCFAs and inosine promote the 
activation and tumor infiltration of CD8+ T cells 
and Th1 cells (Zhao et al., 2025). These 
activated T cells produce IFN-γ, which can have 
complex effects on neutrophils but can also 
contribute to an anti-tumor immune cycle that 
suppresses the pro-tumorigenic functions of 
neutrophils, including excessive NET formation. 
Conversely, a metabolite profile that favors Treg 
expansion and M2 macrophage polarization can 
create an immunosuppressive environment that 
indirectly permits sustained NETosis. 
 
Gut microbial metabolites function as master 
regulators of NETosis through a multi-pronged 
strategy involving direct epigenetic and 
enzymatic interference, receptor-mediated 
signaling, and systemic modulation of 
inflammatory tone. A favorable microbiome 

generates a metabolite profile that acts as a 
"brake" on pathological NETosis, while a 
dysbiotic microbiome often releases the 
"accelerator," contributing to an ICB-resistant 
TME (Saha and Goswami, 2025, Korbecki et al., 
2025). 
 

4. THE GUT-NET-ICB AXIS: AN 
INTEGRATED MODEL 

 
We propose a comprehensive model wherein the 
gut microbiome dictates clinical response to 
Immune Checkpoint Blockade (ICB) through the 
systemic regulation of Neutrophil Extracellular 
Trap (NET) formation, with microbial metabolites 
serving as the primary signaling intermediaries. 
This "Gut-NET-ICB Axis" provides a mechanistic 
framework to explain how the intestinal 
microbiota can influence anti-tumor immunity in 
distant tissues. The model pivots on the balance 
between two contrasting states: a NETosis-
Suppressive, ICB-Permissive state and a 
NETosis-Promotive, ICB-Resistant state. 
 
The following table outlines the contrasting 
characteristics of these two states within our 
integrated model. 
 

4.1 The ICB-Permissive Cycle: A 
Favorable Microbiome Suppresses 
Nets 

 
In this virtuous cycle, a diverse and balanced gut 
microbiome, characterized by taxa such as 
Akkermansia muciniphila, Faecalibacterium 
prausnitzii, and Bifidobacterium spp., generates 
a metabolite profile rich in SCFAs, polyamines, 
and AhR ligands. 
 
Metabolite Production: These microbes 
ferment dietary fiber to produce high levels of 
butyrate and other SCFAs. They also generate 
immunomodulatory polyamines like spermidine 
and tryptophan-derived AhR ligands. 
 
Systemic Signaling: These metabolites are 
absorbed into the portal circulation and exert 
systemic effects. They reach the bone           
marrow and the TME, where they encounter 
neutrophils. 
 

NETosis Suppression: As detailed in Section 4, 
these metabolites directly "raise the threshold" 
for NETosis. Butyrate (via HDACi) and 
spermidine (via PAD4 inhibition) disrupt the core 
biochemical pathways required for chromatin 
decondensation and NET release. 



 
 
 
 

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264 

 

TME Remodeling: The consequent low level of 
NETs in the TME dismantles a major 
immunosuppressive barrier. CD8+ T cells can 
effectively infiltrate the tumor parenchyma, 
encounter their antigen, and execute cytotoxic 
functions without being suppressed by NET-
derived components like histones and proteases. 
 
Effective ICB Response: In this permissive 
environment, the administration of ICB can 
effectively reinvigorate these functional, tumor-
infiltrating T cells, leading to robust tumor control 
and clinical response (Zhao et al., 2016, Mager 
et al., 2020, Xi et al., 2022). 
 

4.2 The ICB-Resistant Cycle: Dysbiosis 
Promotes Net-Driven Immuno-
suppression 

 

Conversely, a state of dysbiosis, potentially 
driven by antibiotics, a Western diet, or disease, 
initiates a vicious cycle that culminates in ICB 
resistance. 
 

Dysbiosis and Metabolite Imbalance: The gut 
community is depleted of beneficial metabolite 
producers. This leads to low systemic levels of 
SCFAs and spermidine. Concurrently, there may 
be an expansion of bacteria that produce pro-

inflammatory metabolites or, through a 
weakened gut barrier, allow the translocation of 
microbial products like Lipopolysaccharide (LPS) 
(Ajibola and Ayodele, 2025). 
 
Loss of NETosis Suppression: The absence of 
the "NETosis brake" (SCFAs, spermidine) leaves 
neutrophils hypersensitive to activation signals. 
Furthermore, circulating LPS and certain 
secondary bile acids provide direct triggers for 
NETosis. 
 
NET-Driven Immunosuppressive TME: 
Neutrophils in the TME readily undergo NETosis, 
leading to high NET density. These NETs 
physically trap T cells and prevent their infiltration 
(Deng et al., 2025). More critically, NET 
components have been shown to directly 
promote T cell exhaustion by upregulating PD-1 
expression and suppressing their proliferative 
and cytotoxic capacity (Cai et al., 2025). 
 
ICB Failure: In this scenario, the TME is 
characterized by excluded and profoundly 
exhausted T cells. Even when PD-1 is blocked 
by ICB, the T cells are too dysfunctional or 
physically sequestered to mount an effective 
anti-tumor response, resulting in primary 
therapeutic resistance. 

 

Table 3. The Two States of the Gut-NET-ICB Axis 
 

Feature NETosis-Suppressive, ICB-
Permissive State 

NETosis-Promotive, ICB-Resistant State 

Gut Microbiome Profile "Favorable" / Eubiotic. Enriched 
with SCFA-producers 
polyamine-producers, and AhR-
ligand generators. 

"Unfavorable" / Dysbiotic. Enriched with 
pathobionts, reduced microbial diversity, and 
bacteria producing pro-inflammatory 
metabolites. 

Systemic Metabolite 
Profile 

High circulating levels of 
Butyrate, Propionate, 
Spermidine, and Indoles. 
Balanced secondary bile acid 
ratio. 

Low levels of beneficial metabolites. High 
levels of pro-inflammatory secondary bile 
acids. 

Impact on Neutrophils & 
NETosis 

Suppressed Pathological 
NETosis. Metabolites directly 
inhibit PAD4, reduce ROS, and 
block HDACs. Neutrophils are 
less prone to excessive NET 
release. 

Promoted Pathological NETosis. Lack of 
suppression and presence of inflammatory 
inducers lead to rampant NET formation. 

Tumor Microenvironment 
(TME) 

-  Low NET Density 
- Enhanced T-cell Infiltration: 
Physical barrier is removed. 
- Functional T-cells: Reduced T-
cell exhaustion and impairment. 
- Favorable CD8+/Treg ratio. 

- High NET Density 
- Impaired T-cell Infiltration: NETs form a 
physical barrier. 
- T-cell Exhaustion: NET components directly 
suppress T-cell function. 
- Immunosuppressive milieu. 

Final ICB Outcome Responsive. Checkpoint 
inhibitors effectively reinvigorate 
pre-existing, functional, and 
infiltrated T cells, leading to 
tumor cell killing. 

Refractory. T cells are excluded, dysfunctional, 
and exhausted; ICB fails to initiate an effective 
anti-tumor response. 



 
 
 
 

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265 

 

 
 

Fig. 2. Acquired resistance to immune checkpoint blockades (Bi et al., 2025) 
 

4.3 Therapeutic Implications of the Axis  
 

This model directly suggests novel therapeutic 
strategies aimed at interrupting the resistant 
cycle and promoting the permissive one. These 
interventions can target different nodes of the 
axis: 
 

Targeting the Gut: Using prebiotics, probiotics, 
or FMT to establish a NETosis-suppressive 
microbiome. 
 

Targeting the Metabolite: Using postbiotic 
supplements to directly deliver the inhibitory 
signals. 
 

Targeting NETosis: Using pharmacological 
PAD4 inhibitors (e.g., GSK484) or DNase I to 
directly prevent NET formation or degrade 
existing NETs, thereby breaking the 
immunosuppressive barrier and potentially 
synergizing with ICB (Bi et al., 2025, Zhang et 
al., 2025). 
 

The Gut-NET-ICB Axis provides a powerful, 
mechanistic model that integrates the fields of 
microbiome research, neutrophil biology, and 
cancer immunotherapy (Zhou et al., 2021). It 
explains how a systemic factor of the gut 
microbiome can locally determine treatment 
efficacy by controlling a specific innate immune 
process, NETosis, that directly shapes the 
adaptive anti-tumor response (Cai et al., 2025, 
Pirini et al., 2025). 
 

5. CONCLUSION 
 

The intricate interplay between the gut 
microbiome, host immunity, and cancer therapy 
continues to redefine our understanding of 

oncobiology. This review has synthesized 
compelling evidence to establish a novel and 
critical pathway: the Gut-NET-ICB Axis. We have 
delineated a mechanistic framework wherein gut 
microbial metabolites serve as pivotal systemic 
regulators of neutrophil extracellular trap (NET) 
formation, which in turn exerts a profound 
influence on the efficacy of immune checkpoint 
blockade (ICB). 
 

The model posits two divergent states. A 
favorable gut microbiome, characterized by 
producers of short-chain fatty acids (SCFAs), 
polyamines, and AhR ligands, generates a 
metabolite profile that systemically suppresses 
pathological NETosis. This creates a tumor 
microenvironment (TME) that is permissive to T-
cell infiltration and function, thereby enabling a 
robust response to ICB. Conversely, a state of 
dysbiosis fails to provide these suppressive 
signals and may even promote NETosis, leading 
to a NET-rich, immunosuppressive TME that 
fosters T-cell exclusion and exhaustion, 
ultimately resulting in ICB resistance. 
 

This paradigm shift from viewing the microbiome 
as a simple correlate to understanding it as an 
active modulator of specific innate immune 
effector mechanisms like NETosis has significant 
implications. It moves the field beyond taxonomic 
associations and towards a functional, 
metabolite-driven understanding of therapy 
response. The axis provides a unifying 
explanation for how a distal community of 
microbes can precisely tune the immune 
landscape of a distant tumor. 
 

Looking forward, this knowledge opens up a 
promising frontier for clinical translation. The Gut-



 
 
 
 

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NET-ICB axis is not merely a explanatory model; 
it is a roadmap for therapeutic innovation. 
Strategies targeting the gut microbiome, its 
metabolic output (postbiotics), or the downstream 
NETotic process itself (PAD4 inhibitors, DNase) 
represent tangible, combinatorial approaches to 
overcome immunotherapy resistance. Future 
research must focus on validating these 
mechanistic links in human cohorts, identifying 
predictive metabolite and NET-based 
biomarkers, and launching clinical trials that 
strategically manipulate this axis. By harnessing 
the power of the gut microbiome to disarm a key 
mechanism of immunosuppression, we can 
ultimately expand the reach and success of 
cancer immunotherapy for a greater number of 
patients. 
 

5.1 Future Perspectives 
 
The elucidated Gut-NET-ICB axis provides a 
powerful, mechanistic framework that connects 
the gut microbiome's metabolic output to a 
specific innate immune process, NETosis, which 
critically determines the efficacy of adaptive 
immunotherapy. This model not only deepens 
our fundamental understanding of ICB resistance 
but also unveils a suite of novel, actionable 
strategies to improve clinical outcomes. Based 
on the evidence synthesized in this review, we 
put forth the following recommendations for 
future research and clinical translation: 
 
1. Prioritize Functional Metabolomics in 
Clinical Cohorts:  Future clinical studies 
correlating the microbiome with ICB response 
must move beyond 16S rRNA sequencing and 
integrate deep metagenomics with metabolomic 
profiling of patient serum and feces. The primary 
goal should be to define a "NETosis-Suppressive 
Metabolite Signature" that can serve as a 
superior predictive biomarker compared to 
taxonomic data alone. 
 
2. Establish Causality in Gnotobiotic Models:  
To move from correlation to causation, well-
defined gnotobiotic mouse models should be 
employed. Colonizing germ-free mice with 
consortia of bacteria engineered to produce 
specific metabolites (e.g., SCFA-producers 
versus non-producers) and subjecting them to 
ICB and NETosis assays will provide direct proof 
of the metabolite-NET link. 
 
3. Explore Combinatorial Clinical Trials:  The 
most immediate translational path involves 
designing clinical trials that combine ICB with 

interventions targeting the Gut-NET axis. We 
recommend prioritizing: 
 
ICB + Pre/Postbiotics: Testing high-fiber diets 
or SCFA/spermidine supplements in conjunction 
with anti-PD-1 therapy. 
 
ICB + NETosis Inhibition: Investigating the 
safety and efficacy of PAD4 inhibitors or DNase I 
in overcoming resistance in selected cancer 
types. 
 
4. Develop Standardized NET Biomarkers: 
The field requires a concerted effort to 
standardize the measurement of NETs in human 
samples. Validated assays for circulating NET 
remnants are needed to correlate systemic NET 
load with metabolite levels and ICB response in 
patient cohorts. 
 
5. Personalize Microbiome-Modulating 
Therapies: Recognizing that a "one-size-fits-all" 
microbiome intervention may be ineffective, we 
recommend developing diagnostic platforms to 
match patients with dysbiotic, NETosis-promoting 
profiles to tailored interventional strategies, such 
as FMT from donors with a validated beneficial 
metabolome. 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 
Author(s) hereby declare that NO generative AI 
technologies such as Large Language Models 
(ChatGPT, COPILOT, etc) and text-to-image 
generators have been used during writing or 
editing of this manuscript.  
 

CONSENT AND ETHICAL APPROVAL 
 
It is not applicable. 
 

COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 
 

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