Treatment with Cistus incanus and Rosmarinus officinalis reduces inflammatory processes in an in vitro virus mimicry approach

Abstract

Acute respiratory viral diseases caused by single-stranded RNA (ssRNA) viruses like influenza A infection occur frequently in all age groups, with significant impact on morbidity and mortality worldwide. The severity and burden of the disease is tightly connected to host inflammatory processes. This study was conducted to investigate immunomodulatory effects of 10 different herbal extracts in human lung epithelial cells (A549) in an in vitro viral mimicry approach that reflects general ssRNA virus recognition. Plant extracts were screened for cytotoxicity on A549 cells. The highest non-toxic herbal doses were further tested on A549 treated with IFN-α and RNA analogues to mimic ssRNA viral infections via the toll-like receptor TLR7/8 activation, thereby establishing an interferon-primed inflammatory state that reflects aspects of dysregulated host response during severe viral respiratory infections. Using flow cytometry and transcriptomics analysis, cytokine release and changes in mRNA expression were analyzed. Non-toxic concentrations of Cistus incanus and Rosmarinus officinalis decreased the secretion of pro-inflammatory cytokines and chemokines e.g. IL-6, IFN-λ1, IL-8, IFN-λ2/3 and IFN-β in stimulated A549 cells. The other eight plant extracts showed no effect in non-toxic concentrations. Through mRNA analyses and Western blotting, we were able to show that NF-κB-related signaling, which plays a key role in driving inflammatory processes during a viral infection, is downregulated by C. incanus and R. officinalis without affecting the protein amount of the anti-viral proteins IFIT2, OASL and TFNAIP3. In addition, a decrease in gene expression associated with cell death could be shown. HPLC analysis revealed that the main compounds in C. incanus extract were ellagitannins and flavonoids, whereas the main constituents in R. officinalis extract were rosmarinic acid and flavonoids. This in vitro virus mimicking model, representing general ssRNA respiratory virus infection, demonstrates that R. officinalis and C. incanus possess promising immunomodulatory and anti-inflammatory properties, supporting their potential as an early treatment option in viral respiratory diseases.