Winner, winner, bovine dinner?

"Distinct innate immune activation profiles of an mRNA-lipid nanoparticle vaccine compared to the empty lipid nanoparticle" by Zelkoski and colleagues

Zelkoski et al.¹ did it! They published a new paper clearly identifying LNPs as the main driver of, uhm, whatever, uh, “immune activation.” Let’s first examine the work in detail:

“Overall, these data corroborate the surface marker data, demonstrating the inherent stimulatory capacity of empty LNPs. However, the mRNA-LNP elicits a more pronounced response by NK cells, T cells and B cells, and increased GM-CSF and IL-10 suggesting that mRNA incorporated with LNP or presentation of spike protein contributes to an increased upregulation of co-stimulatory molecules on these cell subsets and a differential regulation of cytokine production.”

Oh dear. This statement is—and I think Maria Gutschi and I have hopefully elaborated on this exhaustively enough—absolutely untenable: since here the colloidal nature of modRNA-LNPs, and not modRNA + LNP, must first be considered.

The peripheral blood mononuclear cells (PBMCs) came from healthy donors before SARS-CoV-2 exposure and were cryopreserved. The lipid nanoparticles (LNPs) followed the BNT162b2 formulation (ALC-0315, cholesterol, DSPC, ALC-0159 in a ratio of 46.3:42.7:9.4:1.6) and contained messenger RNA (mRNA) for the Omicron spike protein with pseudouridine modification. The thawed peripheral blood cells were cultured in cell culture medium consisting of RPMI 1640 with L-glutamine, 10% heat-inactivated fetal bovine serum, and penicillin-streptomycin. The cells were treated with DNase, incubated for 45 minutes at 37°C, washed, and adjusted to 1 million cells per well. Stimulation was performed with empty lipid nanoparticles (1 μg/mL), fluorescence-labeled lipid nanoparticles (1 μg/mL), mRNA-lipid nanoparticles (1 μg/mL), or R848 (0.25 μg/mL, an immune stimulator) for 24 hours at 37°C and 5% carbon dioxide. After incubation, supernatants were stored at −80°C for cytokine measurements (inflammatory mediators such as interleukin-1β, interleukin-6, tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, interleukin-10). The cells were stained for spectral flow cytometry and analyzed.

Unfortunately, however, this work is also rather geared toward damage control. Or did the authors once again not know better?: Here’s the real hammer that one all too gladly overlooks in 5 pages of hardcore technical jargon in the methods section: “The thawed peripheral blood cells were cultured in cell culture medium consisting of RPMI 1640 with L-glutamine, 10% heat-inactivated fetal bovine serum, and penicillin-streptomycin." Please therefore abandon the idea that a protein corona situation comparable to human serum will develop here in vitro. Please also abandon the idea that you would observe complement system activation here.

Let’s return to the following sentence:

“However, the mRNA-LNP elicits a more pronounced response by NK cells, T cells and B cells, and increased GM-CSF and IL-10 suggesting that mRNA incorporated with LNP or presentation of spike protein contributes to an increased upregulation of co-stimulatory molecules on these cell subsets and a differential regulation of cytokine production.”

Did the authors hope we wouldn’t read further and discuss the following passage?

“While neither LNP nor mRNA-LNP induced substantial production of the classical pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, both induced marked production of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, and interferon (IFN)-β. The magnitude of GM-CSF and IL-10 was significantly greater after cell stimulation with mRNA-LNP, whereas IFN-β was greater after stimulation with LNP alone.”

The first statement deliberately obscures that empty LNPs alone induce GM-CSF and IL-10. Only later do the authors reveal this critical fact—burying it in technical language. And interleukin 10 (IL-10) on top of that? Why is this important? Well… What the authors call an immune response here, I call winning the lottery with strong indication of epigenetic reprogramming with JAK/STAT dysregulation:

Why ‘lottery’? Because this cytokine profile—IL-10 + GM-CSF without pro-inflammatory counterparts—is the molecular signature of epigenetic reprogramming (Zheng et al., 2020²). Not acute immune activation. Not coordinated response. Persistent immune dysregulation.

But step by step: We first look at the work by Riley et al. 1999³ (the good old days when experiments were still done cleanly): “Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action” demonstrates impressively precisely that the interleukin 10-JAK/STAT axis is inseparable, and thus one can factually conclude a JAK/STAT axis shift. Was there something else? Oh yes: interleukin 6 and TNF-alpha didn’t really jump either, right?

“While neither LNP nor mRNA-LNP induced substantial production of the classical pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, both induced marked production of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, and interferon (IFN)-β. The magnitude of GM-CSF and IL-10 was significantly greater after cell stimulation with mRNA-LNP, whereas IFN-β was greater after stimulation with LNP alone.”

“IL-10, IL-6, and TNF-alpha: central factors in the altered cytokine network of uremia--the good, the bad, and the ugly” by Stenvinkel et al.⁴ then reveals to us that there is an AXIS between IL-10, IL-6, and TNF-alpha, while works such as “Targeting the Interleukin-6/Jak/Stat Pathway in Human Malignancies” by Sansone et al.⁵ tell us that precisely these interleukins depend on an intact JAK/STAT1-5 axis and form a homeostatic structure (if one takes the trouble to piece together all the papers). There we are back to my favorite topic: network logic.

Be that as it may, why was GM-CSF so important to me?

Zheng et al. (footnote 2) explicitly emphasize in “Epigenetic Changes Associated With Interleukin-10”:

• IL-10 alters DNA methylation = long-term gene expression changes

• IL-10 triggers histone modifications = stable epigenetic signatures

• IL-10 dysregulates miRNA networks = systemic immune disorders

I think DoorlessCarp🐭 will love the following paragraph of the Zheng et al. paper:

”This suggests that miRNA-4492/IL-10 axis involvement in the Jak/STAT signaling pathway may be a key mechanism for chronic sinusitis with NPs. Hatab et al. found that in patients with advanced liver cancer, 3 consecutive months of curcumin, piperine, and taurine combined treatment could reduce circulating levels of IL-10 and miRNA-21, and patients with high baseline levels of IL-10 and miRNA-21 had a worse overall survival rate. Therefore, changes in serum IL-10 and miRNA-21 expression may be prognostic biomarkers in the treatment of HCC. Finally, miRNA-27a enhances the antibacterial activity of macrophages and inhibits the expression of IL-10, thereby regulating the innate immune response. The relationship between miRNA and IL-10 levels in various diseases is summarized in Figure 3 and Table 2.”

Wasn’t there something?

Seger, Gutschi & Seneff (v1 2025 - v5 2026)⁶:

“Our L-DMD hypothesis implicates that LNPs may also drive the immune system’s initial response through membrane-driven effects of transfected cells, thereby impacting systems beyond immune cells alone. This is further supported by the study of Connors et al., who showed that eLNPs induce activation and maturation of monocyte derived dendritic cells (MDDCs) and also upregulated CD40 expression, which led to recruitment of pro T follicular helper (pro-TFH) cell cytokines, IL-6, IL-12, and IL-21. These findings are also consistent with Amor et al.

Furthermore, Qin et al. showed in mice that these effects have transgenerational immunological consequences. Taken together, these findings indicate that LNP-driven immune activation is not restricted to acute innate immune responses or transient antigen-presentation–linked effects, but can also engage epigenetically mediated regulatory programs, consistent with recent evidence demonstrating short-term and persistent epigenetic memory in innate immune cells following BNT162b2 mRNA vaccination. In this context, recent work by Chytla et al., for example, has highlighted that PI(4,5)P₂ signaling is not confined to the plasma membrane but also operates at the nuclear level, where it contributes to transcriptional regulation and chromatin organisation.”

(Off note: Sadly preprints.org messed up our reference work: We had issues with 12 references and inserted a manual break? (Starts at Ref. number 40 so in the end you have 12 not existent references and a skip up to plus 12 (don’t ask me where and which ones… Narf!)

However and nevertheless: the Zelkoski paper’s main point is clear: LNPs are certainly everything... BUT NOT INERT!

1. Zelkoski, A. E., Mitre, E., Alameh, M.-G., & Malloy, A. M. W. (2025). Distinct innate immune activation profiles of an mRNA-lipid nanoparticle vaccine compared to the empty lipid nanoparticle. Vaccine, 43(1), Article 126562. https://doi.org/10.1016/j.vaccine.2025.126562

2. Zheng, Z., Huang, G., Gao, T., Huang, T., Zou, M., Zou, Y., & Duan, S. (2020). Epigenetic Changes Associated With Interleukin-10. Frontiers in immunology, 11, 1105. https://doi.org/10.3389/fimmu.2020.01105

3. Riley, J. K., Takeda, K., Akira, S., & Schreiber, R. D. (1999). Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action. The Journal of biological chemistry, 274(23), 16513–16521. https://doi.org/10.1074/jbc.274.23.16513

4. Stenvinkel, P., Ketteler, M., Johnson, R. J., Lindholm, B., Pecoits-Filho, R., Riella, M., Heimbürger, O., Cederholm, T., & Girndt, M. (2005). IL-10, IL-6, and TNF-alpha: central factors in the altered cytokine network of uremia--the good, the bad, and the ugly. Kidney international, 67(4), 1216–1233. https://doi.org/10.1111/j.1523-1755.2005.00200.x

5. Sansone, P., & Bromberg, J. (2012). Targeting the interleukin-6/Jak/stat pathway in human malignancies. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 30(9), 1005–1014. https://doi.org/10.1200/JCO.2010.31.8907

6. Seger, F., Gutschi, L. M., & Seneff, S. (2026). Lipid Nanoparticles as Active Biointerfaces: From Membrane Interaction to Systemic Dysregulation. Preprints. https://doi.org/10.20944/preprints202511.0517.v5