M1/M2 Polarization in Zebrafish: miRNA Regulation of M. fortuitum Infection

Last Updated: December 23, 2025
Estimated reading time: ~7 minutes

M1/M2 polarization represents the dynamic plasticity of macrophages, allowing them to switch between pro-inflammatory (killing) and anti-inflammatory (healing) states. This study investigates how the bacterial pathogen Mycobacterium fortuitum exploits or triggers these states in zebrafish kidney macrophages (ZFKM) through specific microRNA switches. By manipulating the expression of miR-155 and miR-146a, the host immune system attempts to balance the elimination of the pathogen with the preservation of host tissue, a process heavily dependent on the resulting macrophage phenotype. Search intent: explain.

Key Takeaways:

• Macrophage Plasticity: ZFKM exhibit distinct M1 (classical) and M2 (alternative) activation states in response to M. fortuitum.
• miR-155 Role: Acts as a driver for M1 polarization, upregulating cytokines like IL-12 and TNF-α to promote bacterial killing.
• miR-146a Role: Drives M2 polarization, enhancing TGF-β and IL-10 expression, which aids bacterial survival.
• Signaling Pathways: The phenotypic switch is mediated through the T-bet/Stat1 axis (M1) and the suppression of NF-κB via IRAK-1/TRAF-6 targeting (M2).
• Bacterial Outcome: The polarization state directly correlates with the intracellular fate of the bacteria (clearance vs. persistence).

To study the role of miRNAs involved in the pathogenesis induced by M. fortuitum in kidney macrophages of zebrafish

miR-155: The Driver of M1 Polarization

The induction of the M1/M2 polarization spectrum begins with the host’s immediate danger response. In the context of M. fortuitum infection, the study identifies miR-155 as the critical molecular switch that locks macrophages into the M1, or classically activated, phenotype. This state is characterized by a robust pro-inflammatory profile designed to obliterate intracellular pathogens. The thesis data indicates that overexpression of miR-155 leads to a significant upregulation of M1 signature genes, specifically Interleukin-1 beta (IL-1β) and Interleukin-12 (IL-12). These cytokines are essential for recruiting other immune cells and creating a hostile environment for the bacteria.

“We observed that miR-155 mimic augmented M1 macrophage function by inducing the expression of IL-1β and IL-12 mRNA and down-regulated the expression of M2 specific TGF-β and IL-10 mRNA expression in M. fortuitum infected ZFKM.” (Mehta, 2021, p. 61)

Mechanistically, this polarization is achieved because miR-155 activates the T-bet/Stat1 signaling pathway. T-bet and Stat1 are transcription factors that are master regulators of the Th1-type immune response, which correlates with M1 macrophage activation. By enhancing the expression of these factors, miR-155 ensures the cell remains in an aggressive, bactericidal state. However, a notable finding in this specific zebrafish model was that while IL-12 and IL-1β were upregulated, inducible Nitric Oxide Synthase (iNOS)—often a hallmark of M1 activation in mammals—was not significantly modulated by miR-155 mimics, suggesting species-specific nuances in the immune response.

Student Note: M1 Macrophages are “classically activated” and are pro-inflammatory and microbicidal; M2 Macrophages are “alternatively activated” and are anti-inflammatory and tissue-repairing.

Professor’s Insight: The uncoupling of iNOS from other M1 markers in zebrafish highlights the importance of using species-specific markers when studying comparative immunology.

miR-146a: Promoting the M2 Phenotype

In contrast to the aggressive M1 state, the M1/M2 polarization balance is tipped toward the M2 phenotype by miR-146a. This miRNA functions as a dampener of inflammation, likely evolved to prevent excessive tissue damage during infection. The research demonstrates that when miR-146a is overexpressed in ZFKM infected with M. fortuitum, there is a marked shift in cytokine production. The cells reduce their output of pro-inflammatory signals and instead upregulate M2 signature genes, particularly Transforming Growth Factor-beta (TGF-β) and Interleukin-10 (IL-10).

“miR-146a on the other hand, augments anti-inflammatory responses (IL-10 and IL-4) and promotes M2 phenotype (TGF-β)…” (Mehta, 2021, p. 1)

This shift is not merely a passive resolution of inflammation but an active reprogramming of the cell. The mechanism involves miR-146a targeting and silencing IRAK-1 and TRAF-6. These proteins are critical transducers in the Toll-Like Receptor (TLR) pathway that normally leads to NF-κB activation and M1 polarization. By severing this signaling link, miR-146a effectively “turns off” the M1 program, allowing the M2 program to take precedence. While this aids in tissue repair, the study found it comes at a significant strategic cost: the bacteria survive and replicate more efficiently within M2-polarized macrophages.

Student Note: TGF-β (Transforming Growth Factor-beta) is a potent immunosuppressive cytokine that is a hallmark of the M2 macrophage phenotype and tissue repair processes.

miRNA	Target Gene(s)	Effect on Phenotype	Cytokine Profile	Bacterial Outcome
miR-155	SOCS1	Promotes M1	↑ TNF-α, IL-12, IFN-γ	Clearance / Apoptosis
miR-146a	IRAK-1, TRAF-6	Promotes M2	↑ TGF-β, IL-10	Survival / Persistence

Fig: Differential regulation of macrophage polarization by miRNAs in M. fortuitum infection (Based on Mehta, 2021, p. 84).

Professor’s Insight: The “M2” state in bacterial infection is often colloquially referred to as “alternatively activated,” but in the context of pathogenesis, it can be viewed as a “permissive” state for the microbe.

The Consequence of Polarization on Bacterial Fate

The ultimate biological relevance of M1/M2 polarization lies in the survival or death of the invading pathogen. The thesis provides compelling evidence linking these miRNA-driven phenotypic switches directly to bactericidal capacity. The M1 state, driven by miR-155, is associated with high rates of apoptosis (programmed cell death) and efficient bacterial clearance. The study utilized Colony Forming Unit (CFU) assays to demonstrate that enhancing miR-155 levels significantly reduced the intracellular bacterial load.

“Using combination of miRNA specific mimic and inhibitor we established that miR-155 induces… M1… phenotype of macrophages… and thus leads to bacterial clearance.” (Mehta, 2021, p. 1)

Conversely, the M2 state driven by miR-146a created a sanctuary for M. fortuitum. By suppressing the oxidative burst (downregulation of iNOS) and inhibiting apoptosis, miR-146a allowed the bacteria to persist and multiply within the macrophages. This highlights a critical “tug-of-war” in host-pathogen interactions: the host strives for an M1 state to clear the infection, while the pathogen may benefit from (or actively trigger) an M2 state to establish a latent or chronic infection. Understanding this balance is crucial for developing therapies that can force the immune system out of a permissive M2 state and into a reactive M1 state.

Student Note: Granulomas in tuberculosis often contain a mix of M1 and M2 macrophages; the ratio can determine whether the granuloma contains the infection or allows it to disseminate.

Professor’s Insight: Therapeutic strategies often aim to “re-polarize” tumor-associated macrophages or infection-associated macrophages from M2 back to M1 to restore immune efficacy.

Reviewed and edited by the Professor of Zoology editorial team. Aside from direct thesis quotations, the content is educational and original.

Real-Life Applications

• Immunomodulatory Therapies: Identifying miR-155 as an M1 driver suggests it could be a candidate for immunotherapy to boost innate immunity against antibiotic-resistant bacteria in aquaculture.
• Disease Biomarkers: The ratio of miR-155 to miR-146a in fish tissues could serve as a diagnostic biomarker to determine the stage of infection (acute/clearing vs. chronic/persistent).
• Vaccine Development: Adjuvants that specifically upregulate miR-155 could enhance the efficacy of fish vaccines by ensuring a strong initial M1 response.
• Zoonotic Risk Assessment: Understanding how M. fortuitum persists in fish macrophages (M2 state) helps assess the risk of transmission to humans, especially in aquaculture workers.

Key Takeaways

• Phenotypic Switching: Macrophages are not static; they actively switch between M1 (killer) and M2 (healer) states during infection.
• Epigenetic Control: This switch is controlled epigenetically by specific miRNAs (miR-155 vs. miR-146a).
• M1 Signature: miR-155 promotes Th1-like responses (IFN-γ, TNF-α) and apoptosis to clear bacteria.
• M2 Signature: miR-146a promotes Th2-like responses (IL-10, TGF-β) which inadvertently aids bacterial survival.
• Molecular Targets: The balance is maintained by targeting SOCS1 (by miR-155) and IRAK-1/TRAF-6 (by miR-146a).

MCQs

1. Which cytokine is identified in the thesis as a hallmark of the M2 macrophage phenotype induced by miR-146a?
   A. TNF-α
   B. IL-12
   C. TGF-β
   D. IFN-γ
   Correct: C
   Explanation: The thesis notes that miR-146a overexpression augments TGF-β and IL-10, which are anti-inflammatory M2 markers (Mehta, 2021, p. 1).
2. In the context of M. fortuitum infection in zebrafish, how does miR-155 affect iNOS expression according to the specific knockdown studies?
   A. Significantly increases iNOS
   B. Significantly decreases iNOS
   C. No observed change in iNOS expression
   D. Completely inhibits iNOS
   Correct: C
   Explanation: The thesis explicitly states, “Interestingly, we did not observe any change in the expression of iNOS when treated with miR-155 mimic and inhibitor” (Mehta, 2021, p. 61).

FAQs

Q: What determines whether a macrophage becomes M1 or M2?
A: The local cytokine environment and specific signaling molecules, such as microRNAs (miR-155 for M1, miR-146a for M2), drive the polarization state.

Q: Why would the host activate M2 macrophages if they help bacteria survive?
A: M2 activation is a negative feedback mechanism intended to prevent excessive tissue damage (immunopathology) caused by prolonged inflammation, but pathogens can exploit this.

Q: Can M. fortuitum induce both phenotypes?
A: Yes, the infection triggers a dynamic response where early M1 activation (clearance) may be followed or counteracted by M2 activation (persistence/regulation).

Lab / Practical Note

When studying macrophage polarization in vitro, ensure that culture media are free of endotoxins (LPS), as even trace amounts can artificially skew macrophages toward the M1 phenotype, confounding results related to M. fortuitum infection.

External Resources

• Macrophage Polarization: Nomenclature and Plasticity (NCBI)
• Zebrafish as a Model for Tuberculosis (ScienceDirect)

Sources & Citations

Title: To study the role of miRNAs involved in the pathogenesis induced by M. fortuitum in kidney macrophages of zebrafish
Researcher: Priyanka Mehta
Guide/Supervisor: Prof. Umesh Rai (Supervisor), Prof. Shibnath Mazumder (Co-supervisor)
University + Location: University of Delhi, Delhi, India
Year: 2021
Pages used: 1, 61-62, 76-78, 84.

Author Box

Priyanka Mehta, PhD Scholar, Department of Zoology, University of Delhi.
Disclaimer: This summary is provided for educational purposes only and does not constitute medical advice.
Reviewer: Abubakar Siddiq, PhD, Zoology
Note: This summary was assisted by AI and verified by a human editor.

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