Photoinduced Inhibition of Neutrophil Extracellular Traps Formation by Dichromatic Light Irradiation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Neutrophils are the first line of defense of the human immune system against pathogens.  Photobiomodulation has emerged as a method to modulate neutrophil function through targeted light exposure. The authors previously reported the effects of monochromatic irradtion on neutrophil extracellular traps (NETs) formation, and in this manuscript, the effects of dichromatic LED-light irradiation on NETs formation were systematically examined. The authors found that combined simultaneous irradiation with 415 nm and 625 nm LEDs resulted in a significant suppression of NETs formation, highlighting a potent inhibitory synergy and suggesting a new approach of wavelength pairing to modulate neutrophil activation. The results delineate a wavelength- and ROS-dependent framework for light-induced neutrophil activation. Overall, this research is interesting, and the writing is also good. I recommend the acceptance of this manuscript.

1. For description of dual wavelengths, “365+625nm”may be unclear, and can be changed to “365nm+625nm ”in the figures.
2. Whether light intensities could influence biological effects? For dual wavelengths, whether the light intensities are the same as the cases of monochromatic wavelengths?
3. Near-infrared light may be also checked, since such light has good deep tissue penetration for applications in vivo.

Author Response

Responses are in the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Neutrophils are central to innate immunity, and their function can be modulated by photobiomodulation through mitochondrial photoacceptors such as cytochrome c oxidase. Although NET formation (NETosis) has been extensively studied, the wavelength-specific regulation of neutrophil photoactivation and its underlying redox mechanisms remain poorly understood. This manuscript provides a clear and original investigation into how mono- and dichromatic light irradiation influence NET formation. The authors demonstrate distinct wavelength-dependent effects: violet-blue light (415 nm) strongly induces NET release via mitochondrial ROS, whereas simultaneous exposure with red light (625 nm) reduces this response. The use of pharmacological inhibitors (apocynin, MitoTEMPO) further clarifies the relative roles of NADPH oxidase and mitochondrial ROS. The study is carefully designed, the figures are well presented, and the discussion effectively links the findings to broader concepts of photobiomodulation and immune regulation. Overall, this work makes a novel and valuable contribution, highlighting dichromatic light exposure as a potential strategy to modulate neutrophil activity and inflammatory responses.

 

Comments:

Figure 1. The legend would benefit from additional details, such as the percentage of cell confluence at the time of irradiation. Including this information will help readers better understand the experimental conditions and improve reproducibility.

Figure 2. Consider adding direct labeling on the representative images (e.g., ‘NETosis’ vs. ‘intact cells’) to guide non-expert readers. This would make the figure more accessible and improve clarity.

Figure 3. The text should provide more detail on the interpretation of the chemiluminescence (CL) signal. Please clarify whether CL intensity is being used as a direct indicator of ROS formation, and if so, specify which ROS species are primarily detected under these conditions.

Figure 4. Under 365 nm irradiation, the apocynin treatment appears more effective than MitoTEMPO in repressing NET formation. Could the authors clarify the mechanistic basis for this difference? For example, does it reflect a stronger contribution of NADPH oxidase–derived ROS relative to mitochondrial ROS at this wavelength?

Why is 415 nm the most effective wavelength for inducing NET formation? Please provide a brief explanation in the text, possibly linking it to absorption characteristics of relevant photoacceptors or mitochondrial chromophores.

The Methods/Results should also briefly explain how PMA promotes NET formation (e.g., by activating PKC and stimulating NADPH oxidase–derived ROS production), so that readers less familiar with neutrophil biology can follow the rationale.

Author Response

Response to the Reviewer

 

1. The legend would benefit from additional details, such as the percentage of cell confluence at the time of irradiation. Including this information will help readers better understand the experimental conditions and improve reproducibility.

 

- Thank you so much for this comment. We added missed information in the legend of Figure 1.

The density of neutrophils in the wells was 2 x 10⁵ cells/mL, i.e. 10⁵ per well. This type of neutrophil seeding allows to obtain a layer of cells in which neutrophils do not contact each other. This avoids unwanted interactions between cells stimulated for NETosis. The percentage of confluence was 30-40% in our experiments.

 

2. Consider adding direct labeling on the representative images (e.g., ‘NETosis’ vs. ‘intact cells’) to guide non-expert readers. This would make the figure more accessible and improve clarity.

 

- Thank you so much for this comment. We corrected Figure 2 with this comment.

 

3. The text should provide more detail on the interpretation of the chemiluminescence (CL) signal. Please clarify whether CL intensity is being used as a direct indicator of ROS formation, and if so, specify which ROS species are primarily detected under these conditions.

 

- Thank you for your comment. The Discussion section has been updated with an additional interpretation of the chemiluminescence.

 

4. Under 365 nm irradiation, the apocynin treatment appears more effective than MitoTEMPO in repressing NET formation. Could the authors clarify the mechanistic basis for this difference? For example, does it reflect a stronger contribution of NADPH oxidase–derived ROS relative to mitochondrial ROS at this wavelength?

- Thank you for this comment. Figure 4 clearly demonstrates the validity of your assumption. Indeed, the photoactivation of NETosis using either apocynin or MitoTEMPO has a different inhibitory effect depending on the wavelength of irradiation: MitoTEMPO inhibits NETosis much more effectively when activated at a wavelength of 415 nm (mitochondrial ROS) than at 365 nm (NADPH oxidase–derived ROS). The opposite picture is true of apocynin, which we have repeatedly observed, including in our first publication on photonetosis [22].

 

5. Why is 415 nm the most effective wavelength for inducing NET formation? Please provide a brief explanation in the text, possibly linking it to absorption characteristics of relevant photoacceptors or mitochondrial chromophores.

 

- Thank you. Information regarding the absorption band of cytochrome c oxidase known as the Soret band, has been appended to the text. This specific absorption band is located within the range of (405-420) nm.

6. The Methods/Results should also briefly explain how PMA promotes NET formation (e.g., by activating PKC and stimulating NADPH oxidase–derived ROS production), so that readers less familiar with neutrophil biology can follow the rationale.

 

- Thank you. We included missed information to the legend of Figure 2 and Methods.