Cyanobacteria are gram-negative photosynthetic prokaryotes, with an estimated 8,000 species, and LPS is a defining feature of their outer membrane. These organisms thrive in freshwater and saltwater environments as well as on damp surfaces, increasing the likelihood of human exposure. Cyanobacterial RNA has been detected in the lungs of people living near contaminated freshwater sources, and cyanobacterial LPS is aerosolized and inhaled by those same individuals. Because LPS composition varies considerably across cyanobacterial species and different preparations show different potencies in provoking human disease, the research team set out to determine whether the inflammatory capacity observed with Geitlerinema sp. LPS would hold for a phylogenetically distinct cyanobacterium. Geitlerinema sp. belongs to the order Oscillatoriales, while Brasilonema sp. belongs to the Nostocales order, making them suitable candidates for testing generality across lineages.

The researchers purified LPS from the HT-58-2 strain of Brasilonema sp. using a hot phenol-water extraction protocol, assessed purity by UV spectrophotometry and SDS-PAGE with a Pro-Q Emerald 300 lipopolysaccharide gel stain, and confirmed biological activity relative to E. coli LPS using a Limulus Amebocyte Lysate endotoxin assay. In the in vivo experiments, C57Bl/6 mice were exposed intranasally to 1 µg/g of either E. coli LPS or Brasilonema sp. LPS, or to phosphate-buffered saline alone, under isoflurane anesthesia. Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue were collected for analysis by flow cytometry, Diff-Quik staining, multiplex cytokine assays, and hematoxylin and eosin histopathology scored by a blinded pathologist.

The results showed that both E. coli and Brasilonema sp. LPS induced lung inflammation visible on histology, with significantly elevated pathology scores compared to PBS-treated controls. Flow cytometry of BALF revealed that both LPS types promoted infiltration of CD11b+Ly-6G+SiglecF- neutrophils, while Diff-Quik staining confirmed increased neutrophil numbers with diminished macrophages, consistent with previously described LPS-induced lung inflammation. Multiplex analysis of BALF demonstrated that Brasilonema sp. LPS enhanced production of the pro-inflammatory cytokine IL-6 and the neutrophil-recruiting chemokine KC (IL-8), without promoting the anti-inflammatory cytokine IL-10. Taken together, these data indicated that Brasilonema sp. LPS promotes lung inflammation in a manner similar to other cyanobacterial species previously studied.

The team then asked whether Brasilonema sp. LPS could exacerbate Th2-dependent allergic airway inflammation, a question that had not been addressed for cyanobacterial LPS before. They used an established ovalbumin (OVA) mouse model of LPS-driven allergic airway exacerbation. Mice were sensitized intraperitoneally with OVA adsorbed to alum adjuvant on day zero, challenged intranasally with OVA on days 13 through 16, and then exposed to low concentrations of either E. coli LPS or Brasilonema sp. LPS on days 22 through 24. Lung histology and BALF were analyzed 24 hours after the final LPS exposure, with pathological scoring performed by a pathologist blinded to treatment group.

In this exacerbation model, OVA-sensitized mice displayed increased lung inflammation and elevated eosinophil infiltration as expected. When E. coli LPS was added, the lungs showed increased inflammation and a mixed infiltrate of both eosinophils from the OVA response and neutrophils from the LPS exposure. Surprisingly, when Brasilonema sp. LPS was added instead, the lungs showed increased inflammation on histology and eosinophil infiltration, but without the additional recruitment of neutrophils as determined by flow cytometry. The researchers noted that low levels of both E. coli and Brasilonema sp. LPS over multiple days promoted some level of lung pathology independent of neutrophil infiltration, likely because the LPS dose used in the exacerbation phase (2.5 ng per mouse) was far lower than the dose that induced neutrophil accumulation alone (1 µg/g in the single-exposure experiments).

To probe the cytokine microenvironment further, the team measured BALF cytokine levels 24 hours after the final LPS exposure. The OVA model induced IL-4 production in all OVA-exposed mice, and OVA-specific IgG1 and IgE were elevated across all OVA-sensitized groups. E. coli LPS added to OVA-sensitized mice increased the presence of Th1/Th17 cytokines, specifically IFN-gamma and IL-17, which are associated with a mixed infiltrate of neutrophils and eosinophils. In contrast, Brasilonema sp. LPS did not influence Th1/Th17 cytokines but did increase TNF-α and RANTES in the BALF of OVA-sensitized mice, suggesting that these low levels of Brasilonema sp. LPS can promote low-level inflammation without recruiting neutrophils. The researchers repeated the exacerbation studies with a lower OVA sensitization concentration (0.1 mg/mL instead of 10 mg/mL) and found the same pattern: eosinophilic infiltration in all OVA-sensitized groups, but neutrophil exacerbation only in mice exposed to E. coli LPS, whether or not OVA was present.

To determine whether Brasilonema sp. LPS signals through TLR4 or through an alternative receptor, the researchers turned first to an in vitro system using the A549 human lung adenocarcinoma epithelial cell line. Brasilonema sp. LPS significantly increased both IL-6 and IL-8 production, and the addition of the TLR4-specific antagonist SPA-4 blocked the induction of both mediators in a concentration-dependent manner without significantly impacting cell viability at 1 and 10 µM, with some impact at 100 µM. The team then confirmed the requirement for TLR4 in vivo by comparing wild-type C57Bl/6 mice with TLR4-deficient (TLR4−/−) mice exposed to either PBS, E. coli LPS, or Brasilonema sp. LPS. Wild-type mice exposed to either form of LPS demonstrated lung inflammation and pathology compared to PBS controls, but this response was absent in TLR4-deficient mice. BALF from TLR4-deficient mice exposed to Brasilonema sp. LPS contained no infiltrating neutrophils by flow cytometry or Diff-Quik analysis, although some residual neutrophil infiltration persisted in TLR4-deficient mice exposed to E. coli LPS, suggesting that E. coli LPS may engage additional pathways. IL-6 and KC (IL-8) remained at baseline levels in TLR4-deficient mice after Brasilonema sp. LPS exposure, while all other cytokines analyzed were unchanged in both wild-type and TLR4-deficient animals.

The authors propose that the neutrophil-independent pathology seen with Brasilonema sp. LPS in the Th2 exacerbation model may arise from TLR4 stimulation within the lung tissue itself, inducing localized edema and cellular retention that is captured in the pathology scoring but not reflected in BALF cell counts. They note that LPS-mediated stimulation of endothelial cells is known to contribute to lung pathology through edema and changes in adhesion molecule expression, and that IL-6, which Brasilonema sp. LPS specifically stimulates A549 cells to produce, is a significant promoter of vascular permeability that results in airway edema. The concentration of Brasilonema sp. LPS used in the exacerbation model was substantially lower than the concentration that induced neutrophil accumulation alone, and the authors acknowledge that higher doses might recruit neutrophils in addition to the pathology seen in the eosinophilic model, though they did not perform those experiments because an equivalent dose of E. coli LPS would be lethal to the mice.

The study has limitations that the authors acknowledge. The exacerbation model was limited to three days of Brasilonema sp. LPS exposure, whereas humans are likely chronically exposed to cyanobacteria-tainted water and aerosols, and it remains possible that chronic exposure could generate a mixed cellular infiltrate that is more challenging to treat than a purely Th2-dependent response. Additionally, while some reports indicate that cyanobacterial LPS from freshwater blooms can be as potent as E. coli LPS, the Brasilonema sp. preparation used here was consistently a weaker stimulus for immune cell activation than E. coli LPS, consistent with the team’s previous findings with Geitlerinema sp. The broader implications are significant: cyanobacterial LPS has been shown to activate human lung carcinoma cells, monocytes and macrophages, peripheral blood mononuclear cells, keratinocytes, intestinal epithelial cells, murine B cells, rat microglia, and fish intestinal cells across multiple studies. As cyanobacterial blooms increase due to global warming and other environmental factors, the authors suggest that TLR4 antagonists such as SPA-4, which have been well tolerated in animal studies and can block E. coli LPS activity in vivo with minimal toxicity, may offer a foundation for future protocols to treat lung inflammation near cyanobacterial blooms, while also raising awareness of the need to monitor and treat water systems effectively.

Subject of Research: Cyanobacterial lipopolysaccharide-induced lung inflammation and TLR4-dependent exacerbation of allergic airway disease in mice

Article Title: Cyanobacterium Brasilonema sp. LPS Induces Neutrophil Infiltration and Lung Inflammation in a TLR4‐Dependent Manner and Exacerbates Th2‐Dependent Lung Inflammation

Article References: Swartzendruber, J., Toro, R. M. D., Alfano, P., Incrocci, R., Volin, M. V., Jiang, K., Mayer, A. M. S., Williams, P. G., O'Donnell, T. J., & Swanson‐Mungerson, M. (2026). Cyanobacterium Brasilonema sp. LPS Induces Neutrophil Infiltration and Lung Inflammation in a TLR4‐Dependent Manner and Exacerbates Th2‐Dependent Lung Inflammation. Immunity, Inflammation and Disease, 14(10), Article e70560. https://doi.org/10.1002/iid3.70560

Image Credits: AI Generated

DOI: 10.1002/iid3.70560

Keywords: cyanobacteria, lipopolysaccharide, TLR4, lung inflammation, neutrophils, Th2 inflammation, asthma, allergic airway disease, Brasilonema, ovalbumin mouse model, SPA-4 antagonist, cytokines