Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE)

Purpose: Nucleoside-modified mRNA lipid nanoparticle (modmRNA-LNP) technology is clinically successful, but its behavior in pre-existing inflammatory or immune-challenged conditions is poorly characterized. This gap raises the risk of adverse effects when modmRNA-LNPs are administered to patients with ongoing inflammation.

Hypothesis: In mice with pre-existing LPS-induced inflammation, IV administration of modmRNA-LNP will exacerbate pro-inflammatory cytokine responses (inflammation exacerbation, IE) in an LNP-dependent, mRNA-cargo-independent, TLR4/macrophage-dependent, and dose/time-dependent manner, and this effect can be mitigated by anti-inflammatory drugs or by temporal/spatial separation of the inflammatory stimulus and LNP.

Aims: Induce acute inflammation in mice with LPS (IT or IV) and then administer modmRNA-LNP IV; screen for inflammatory markers such as IL-6 and MIP-2. - Determine whether IE is specific to the LNP, independent of mRNA cargo, and dependent on LNP formulation/ionizable lipid. - Investigate mechanism using TLR3 KO, TLR4 KO, and macrophage-depleted mice. - Test mitigation strategies: corticosteroids, pioglitazone, temporal separation, and spatial targeting away from liver/spleen. - Assess generalizability to non-LPS inflammatory models (intrastriatal TNF-α and IV oleic acid).

Delivery system: Platform: Nucleoside-modified mRNA lipid nanoparticle (modmRNA-LNP). - LNP composition: Ionizable cationic lipid (proprietary to Acuitas), phosphatidylcholine, cholesterol, PEG-lipid; ~80 nm diameter. - mRNA: m1Ψ-modified, purified mRNA encoding luciferase or other cargo; CpG/dsRNA contaminants removed. - Comparators: Empty-LNP; DLin-MC3-DMA-LNP; C12-200-LNP; LNP without ionizable lipid; liposomes; nanogels; AAV empty capsids. - Targeted LNPs: Anti-PECAM, anti-PLVAP, and anti-VCAM antibody-conjugated LNPs for lung or brain endothelial targeting. - Payload/dose: modmRNA-LNP at 0.032 or 0.32 mg mRNA/kg IV. - Inflammation trigger: LPS from E. coli O111:B4; IV 2 mg/kg or IT 1 mg/kg.

Approach: Mice: C57BL/6J wild-type, TLR3 KO, TLR4 KO; 6–8 weeks old. - Models: LPS-induced systemic inflammation (IV), LPS-induced acute lung inflammation (IT), intrastriatal TNF-α brain inflammation, IV oleic acid acute lung injury. - Timing: LPS at t = 0 h; modmRNA-LNP at t = 4 h unless otherwise stated; tissues harvested at t = 8 h. - Groups/controls: Naive vs LPS-treated; modmRNA-LNP vs empty-LNP vs no treatment; ± clodronate liposomes; ± dexamethasone or pioglitazone; n = 3–6 animals per group. - Doses: LPS 2 mg/kg IV or 1 mg/kg IT; modmRNA-LNP 0.32 mg mRNA/kg IV; clodronate liposomes 50 mg/kg IV; dexamethasone 2 mg/kg IP or SC.

Key methods: ELISA for IL-6 in serum and MIP-2 in liver/brain homogenates. - LEGENDplex mouse inflammation panel for cytokine screening. - qRT-PCR for MIP-2, TLR3, and TLR4 expression. - Bioluminescence imaging (IVIS) for luciferase mRNA translation in liver and spleen. - Flow cytometry for LNP uptake in lung endothelial cells, monocytes/macrophages, and other leukocytes. - Bronchoalveolar lavage (BAL) analysis for leukocyte count and protein concentration.

Key results: IE with LPS + modmRNA-LNP: Serum IL-6 increased 14-fold vs LPS alone and 94-fold vs modmRNA-LNP in naive mice. Liver MIP-2 increased 11-fold vs LPS alone and 52-fold vs modmRNA-LNP in naive mice. Liver MIP-2 mRNA increased 11-fold and 8-fold, respectively. - LNP-specific and mRNA-independent: Empty-LNP caused the same IE; DLin-MC3-DMA-LNP and C12-200-LNP showed similar IE. LNP without ionizable lipid, liposomes, nanogels, and AAV empty capsids did not cause IE. - Reduced mRNA translation: LPS pretreatment reduced luciferase expression ~4-fold in liver and ~3.3-fold in spleen. - Macrophage/TLR4 dependence: LPS increased monocyte/macrophage LNP uptake from ~20% to ~60%. Clodronate depletion reduced IE 10-fold for serum IL-6 and 8-fold for liver MIP-2. TLR4 KO mice showed no IE; TLR3 KO mice behaved like WT. - Mitigation: Dexamethasone reduced IL-6 by 3.5–4-fold; pioglitazone showed partial reduction. IE was lost when modmRNA-LNP was given 24 h after LPS. Lung-targeted anti-PECAM or anti-PLVAP LNPs with systemic LPS showed significantly lower systemic IL-6 and liver MIP-2. - Other inflammatory models: No IE observed with intrastriatal TNF-α or IV oleic acid.

Interpretation: The authors claim this is the first identification of LNP-mediated inflammation exacerbation (IE) in the setting of LPS-induced inflammation. They conclude that IE is LNP-specific, independent of mRNA cargo, macrophage/TLR4-dependent, dose- and time-dependent, and can be partially alleviated by corticosteroids or by temporal/spatial separation. This has implications for using modmRNA-LNPs in patients with gram-negative bacterial inflammation, sepsis, or other TLR4-driven inflammatory states.

Limitations: IE was demonstrated only with LPS-induced inflammation; it did not generalize to TNF-α or oleic acid models. - Findings are limited to mouse models; no large-animal or human validation. - Chronic inflammatory or immune-challenged conditions were not addressed. - The modmRNA-LNP dose used was higher than typical SARS-CoV-2 vaccine doses but lower than some therapeutic mRNA antibody doses. - Mechanistic details beyond TLR4/macrophage involvement remain incompletely defined.