The Underlying Mechanisms Of Monocytes And Pulmonary Interstitial Macrophages’ Trans-differentiation And Function In Ricin Inhaled Severe Pneumonia

Ricin toxin（RT）,derived from castor beans,is highly toxic when inhaled,leading to acute lung injury（ALI）and high mortality risk.RT has terminal mannose residues that bind specifically to mannose receptors on the surface of macrophages,which are early targets of RT.Effects on monocytes and alveolar macrophages（AMs）after RT-induced ALI is relatively well understood.However,while research on the heterogeneity of lung interstitial macrophages（IMs）has progressed,the functional divisions of IMs subpopulations remains to be clarified.Growth differentiation factor 15（GDF15）is a member of the TGF-βsuperfamily and provides protection from against diverse group of inflammatory diseases.Previous studies from our laboratory demonstrated that the transcription level of the Gdf15 gene significantly increases in the early stage of RT-induced ALI（4 h after RT exposure）.Yet,the role of Gdf15 in ALI and whether signaling affects macrophages development and function has not been evaluated.Here,we explore the transdifferentiation of monocytes to IMs,describe the heterogeneity of the resulting IMs and search for key factors affecting the process of cell transdifferentiation.We further investigate the effects of the key factor Gdf15 on the production of monocyte-derived IMs and provide a theoretical basis for further study of laboratory detection markers and therapeutic targets for RT-induced ALI.We challenged C57/BL6 wild-type（WT）mice with 1.5×LD₅₀（lethal dose for 50%of exposed animals）of RT by aerosolized intratracheal inoculation.The dynamics of monocytes and macrophages abundance in lungs after RT exposure,along with the expression of key marker proteins in different cell subpopulations,were then analyzed by conventional flow cytometry combined with mass spectrometry.Subsequently,monocytes and macrophages were separated using flow cytometry and single-cell transcriptomic assays performed at different times post-RT exposure to reveal the transdifferentiation of monocytes to IMs and to quantify IMs cellular heterogeneity.Finally,we used parabiosis and adoptive transfer experiments to reveal the source of regenerating IMs.At steady state,differentiation of lung tissue IMs involves recruiting monocytes from circulation to maintain homeostasis.When lung tissue is exposed to RT,classical and patrolling monocytes are replenished from the circulation and transformed to IMs,creating three subtypes of IMs:IM1s with pro-inflammatory and phagocytic effects;IMregs with pro-inflammatory and immunosuppressive effects;and IM2s with tissue remodeling effects.IM2s subsequently transformed into IM1s and IMregs,further enhancing the phagocytic and regulatory abilities of IMs and thereby maximizing toxin clearance and suppression of excessive immune response.Together,these results reveal the transdifferentiation process of monocytes to IMs and the mechanisms of balance among the three cell subpopulations.In addition,we found that Gdf15 had an important effect on monocytes/IMs transdifferentiation.We thus investigated the intrinsic role of Gdf15 in ALI induced by intratracheal inoculation of a 1.5×LD₅₀ of aerosolized RT in Gdf15 knock-out（KO）compared to C57/BL6 WT mice.KO mice had increased lung tissue pathology,decreased body weights and survival rates,and increased expression of inflammatory-related cytokine and chemokine levels at 24 and 72 h post-RT exposure.Next,we used flow cytometry to quantify the infiltration of myeloid cells in lung tissue post-exposure.Although a similar infiltration pattern was observed in Gdf15 KO and WT groups,KO mice had elevated levels of neutrophils and decreased levels of Ly6C^lomonocytes（cells with distinct destructive and protective roles,respectively）in the early stages of ALI.Gene expression profiles using RNA sequencing and bioinformatic analysis confirmed that pro-inflammatory signaling pathways were activated and that the expression of inflammatory genes was significantly upregulated post-RT exposure compared to corresponding baseline controls in KO and WT mice.To further reveal the effect of Gdf15 on transdifferentiation of monocytes to IMs,we separated monocytes and IMs by flow cytometry to allow single-cell transcriptomic assays and analysis at different times post-RT exposure.After RT exposure,Gdf15 influenced the transcriptional activity of differentiation genes and oxidative phosphorylation to inhibit the transdifferentiation process of monocytes to IMs.Collectively,this study elucidates the transdifferentiation of monocytes to IMs after RT exposure and the resulting heterogeneity of IM subpopulations.This systematic evaluation of immune response signatures in lung tissues reveals the protective effect of Gdf15 in this ALI mice model,deepening our understanding of the relationship and transformation between monocytes and IMs.In addition,our findings expose novel opportunities to investigate the potential role of Gdf15 as a method for treatment of inflammatory lung diseases.