Glyphosate (GLY), as the active ingredient of the most widely used herbicide worldwide, is commonly detected in the environment and living organisms, including humans. Its toxicity and carcinogenicity in mammals remain controversial. Several studies have demonstrated the hepatotoxicity of GLY; however, the underlying cellular and molecular mechanisms are still largely unknown.
Using single-cell RNA sequencing (scRNA-seq), immunofluorescent staining, and in vivo animal studies, we analyzed the liver tissues from untreated and GLY-treated mice.
We generated the first scRNA-seq atlas of GLY-exposed mouse liver. GLY induced varied cell composition, shared or cell-type-specific transcriptional alterations, and dysregulated cell-cell communication and thus exerted hepatotoxicity effects. The oxidative stress and inflammatory response were commonly upregulated in several cell types. We also observed activation and upregulated phagocytosis in macrophages, as well as proliferation and extracellular matrix overproduction in hepatic stellate cells.
Our study provides a comprehensive single-cell transcriptional picture of the toxic effect of GLY in the liver, which offers novel insights into the molecular mechanisms of the GLY-associated hepatotoxicity.
© 2023. The Author(s).

Myofibroblasts are a main cell-type of collagen-producing cells during tissue fibrosis, but their origins remains controversial. While bone marrow-derived myofibroblasts in renal fibrosis has been reported, the cell origin and mechanisms regulating their transition into myofibroblasts remain undefined. In the present study, cell lineage tracing studies by adoptive transfer of GFP+ or dye-labelled macrophages identified that monocyte/macrophages from bone marrow can give rise to myofibroblasts via the process of macrophage-myofibroblast transition (MMT) in a mouse model of unilateral ureteric obstruction. The MMT cells were a major source of collagen-producing fibroblasts in the fibrosing kidney, accounting for more than 60% of α-SMA+ myofibroblasts. The MMT process occurred predominantly within M2-type macrophages and was regulated by TGF-β/Smad3 signalling as deletion of Smad3 in the bone marrow compartment of GFP+ chimeric mice prevented the M2 macrophage transition into the MMT cells and progressive renal fibrosis. In vitro studies in Smad3 null bone marrow macrophages also showed that Smad3 was required for TGF-β1-induced MMT and collagen production. In conclusion, we have demonstrated that bone marrow-derived fibroblasts originate from the monocyte/macrophage population via a process of MMT. This process contributes to progressive renal tissue fibrosis and is regulated by TGF-β/Smad3 signalling.

Inflammation can be prevented in most inflammatory brain diseases, while tissue repair of the lesioned central nervous system (CNS) is still a major challenge. The CNS is difficult to access for protein therapeutics due to the blood-brain barrier. Here, we show that genetically engineered embryonic stem cell-derived microglia (ESdM) are a suitable therapeutic vehicle for neurotrophin-3 (NT3) in experimental autoimmune encephalomyelitis (EAE). The intravenously transplanted ESdM migrated into the inflammatory CNS lesions and engrafted there as microglial cells. EAE afflicted mice treated with ESdM that were genetically modified to express NT3 showed stable recovery from disease symptoms. The NT3-transduced ESdM created an anti-inflammatory cytokine milieu in the spinal cord and promoted neuronal sprouting. Furthermore, mice treated with NT3-transduced ESdM showed less axonal injury and reduced demyelination. Thus, genetically modified ESdM represent a suitable tool to introduce therapeutic neuroprotective and repair-promoting proteins into the CNS in neuroinflammatory diseases.

Monocyte/macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX(3)CR1 is an essential regulator of monocyte/macrophage derived smooth muscle cell differentiation in the vessel wall after injury. Here we hypothesised the contribution of CX(3)CR1- CX(3)CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX(3)CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX(3)CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX(3)CL1-CX(3)CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX(3)CR1 system in vivo, Matrigel plugs were implanted in competent CX(3)CR1(+/gfp) and functionally deficient CX(3)CR1(gfp/gfp) mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX(3)CR1(gfp/gfp) but not in CX(3)CR1(+/gfp) mice. In experimental plaque neovascularization immature MV phenotype was observed in CX(3)CR1(gfp/gfp) mice, lacking CX(3)CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX(3)CR1(+/gfp) mice. This was associated with increased extravasation of platelets into the intima of CX(3)CR1(gfp/gfp) but not functionally competent CX(3)CR1 mice. Pharmacologic targeting using CX(3)CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX(3)CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype.

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Ms) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal Mϕs to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1(+)MHC class II(int)F4/80(hi)CD11b(hi) Mϕs as the main producers of IL-10 via αvβ1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by Mϕs during interactions between IECs and Mϕs. Furthermore, we determined that the administration of recombinant Sema7A proteins ameliorated the severity of colitis, and these effects were diminished by IL-10-blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through αvβ1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mϕs.