17 jan. 2025
Bikash Mishra et al. (BioRxiv)
Keywords
IL-10 mediated suppression
LPS induced inflammation
Human monocytes
Main Findings
Interleukin-10 (IL-10) is a well-recognized immuno-suppressing cytokine, which has been described to inhibit induction of inflammatory cytokines encoded by NF-κB target genes (e.g. TNF, IL6, IL1B and IL12B) in myeloid cells like monocytes and macrophages, as well as dendritic cells. After binding to the IL-10 receptor, the downstream signalling pathway is mediated by the transcription factor STAT3. However, since STAT3 does not directly suppress the targeted inflammatory genes, the exact mechanisms of IL-10 mediated immune-suppression are not clearly understood.
In this preprint, the authors focus on the effects of IL-10 mediated suppression of LPS- induced inflammation in human monocytes. In the main experiments, human blood monocytes are incubated in vitro with IL-10 for 18 hours prior to LPS-stimulation for 3 hours. Another setup also investigates the effects of the 18-hour IL-10 preincubation on subsequent stimulation with TNF (for 6h hours). The effects of IL-10 mediated suppression are analysed on a transcriptomic and epigenetic level by different methods such as RNAseq, ATACseq and CUT&RUN sequencing.
Interestingly, the authors find that rather than suppressing NF-kB and MAPK signalling, IL-10 targets Interferon regulatory factors IRF1 and IRF5 and their DNA binding/transcription factor activity, which further leads to a near-complete suppression of Interferon stimulated genes (ISGs), more than inflammatory genes. These changes are mediated by epigenetic mechanisms such as reduction of chromatin accessibility and de-novo enhancer formation, as well as reduction of IRF1-associated H3K27ac activating histone marks at both ISG and inflammatory gene loci. Similar results to LPS stimulated monocytes were also found in TNF stimulation. Therefore, this study discovered a novel mechanism of IL-10 mediated suppression of inflammatory gene responses after LPS and TNF stimulation in human blood monocytes.
Limitations & Suggestions
Clarification and rationale of IL-10 incubation times and setting.
This study shows that incubation of IL-10 prior to LPS stimulation leads to downregulation of interferon and inflammatory pathways in an in-vitro model using human monocytes. However, it is not fully clear, why specific protocols or stimulation times were chosen (e.g. IL-10 incubation period of 18 hours, 3 hours of LPS treatment, 6 hours of TNF treatment). Since LPS can even induce IL-10, a physiologically more likely setting seems to be that monocytes get in contact with LPS and then stimulated by IL-10. Also, it would be very interesting to understand, how the authors chose the used timepoint of 3 hours post LPS stimulation. Suggestion: Maybe there is already available data from dose testing experiments and different time points of pre-treatment or stimulation, that would be very interesting to include in the study. Otherwise, literature indicating similar protocols could be cited more extensively to increase the understanding of the reader.Applicability of the findings to other stimuli. The authors mainly used LPS stimulation to investigate IL-10 suppression on TLR4 induced inflammation and TNF stimulation to underline their findings in a MAPK/ NF-κB inducing stimulus, however it would be interesting to understand if their findings are specific to these pathways or also hold true other stimulations, especially considering that ISGs can be induced by a plethora of stimuli. Suggestion: Using other stimuli (e.g. INFγ, but also different TLR ligands) after IL-10 incubation would help to get more insight on the pathways described by the authors.
Mechanism and downstream pathway of IL-10 mediated suppression. While the authors find epigenetic alternations and IRF1/IRF5 mediated effects as crucial for IL-10 mediated suppression of inflammatory responses, the exact mechanism downstream of IL-10 receptor binding remains unclear. How does IL-10 lead to these epigenetic changes? The authors mention metabolic reprogramming as a potential mechanism in their introduction, but this is not investigated further. Suggestion: Metabolomics of the IL-10 suppressed monocytes or other metabolic readouts would be very interesting to investigate the impact of metabolic epigenetic reprogramming. Furthermore, the authors could speculate about other potential mechanisms in their discussion and maybe try out methods such as co-immunoprecipitation of the IRFs with or without IL-10 incubation to identify associated proteins that are modulated by IL-10 treatment.
Investigation of findings in a relevant perturbed setting. The primary human monocytes that are investigated in this study are derived from healthy subjects. Interestingly, as the authors state in their discussion, elevated IRF1 levels have been found in different autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus, diseases which can also be accompanied by IL-10 deficiency. Furthermore, IL-10 production is also dysregulated in aging and has been proposed to be important for limiting age associated increased inflammatory effects (“inflammaging”). While the effects of age of the healthy donors is not mentioned in this study, it would be very relevant to further investigate if the study´s findings are changed in older monocytes. Suggestion: While this might be not feasible to include in the same study, monocytes from patients with the stated diseases or patients of different age groups could be compared for chromatin alternation and transcriptional changes upon IL-10 induction, as this would also link the findings to a more clinically relevant setting.
Minor inconsistencies. There is a “with” missing in row 199 (“associated with decreased…”); Fig. 4f: The labelling of the IGF gene tracks is not fully aligned and makes this figure hard to understand/decipher. Also, the last line (IL10+LPS) appears to be cut off; Extended data Fig 1g: In the TNF model experimental setup, the labelling of the TNF only groups still has an LPS written next to it, which should be exchanged to TNF.
Significance/Novelty
In this preprint, the authors used various innovative methods such as RNAseq, ATACseq and CUT& RUNseq to show compelling and well-presented evidence that IL-10 leads to changes chromatin accessibility and downregulation of interferon responses by suppressing IRF1 and IRF5-induction, while the Nf-κB-mediated inflammatory response is mainly not targeted by IL-10. While this is novel and impressive data, the exact mechanisms of IL-10 mediated suppression are not fully clear from this study.
The authors investigate human blood monocytes, which play an important role in a variety of diseases, so more knowledge on their activation and regulation is key for understanding the pathologies in which they are involved. However, the setup used by the study is not physiologically relevant, so further studies with different stimuli and different conditions are needed to fully understand the relevance of the results.
Credit
Reviewed by Martin Murauer and Lisabeth Pimenov as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the Medical University of Vienna and other life science institutes in Vienna.
The authors declare no conflict of interests in relation to their involvement in the review.