CD45⁺Col⁺ cells migrate to spleen in response to CCl₄. a Outline of the experiment: lethally irradiated wild-type mice are transplanted with Col-GFP⁺ BM, and 2 months later subjected to liver injury by CCl₄. CD45⁺Col⁺ were not detected in the liver or spleen of Col-into-wt mice without injury or stress (n=15), upper panel. The organs populated by CD45⁺Col⁺ cells in Col-into-wt mice in response to CCl₄-induced liver injury are liver, gall bladder (GB), spleen, mesentery lymph nodes (LN), and omentum majus (OM), as analyzed by fluorescent microscopy and H&E staining, lower panel (n=25). Scale bars represent 50 μm. b Col⁺ cells co-express CD45, as detected by immunofluorescence or flow cytometry analysis. In response to CCl₄, the number of CD45⁺Col⁺ cells reached 1.3% in the BM and 4.1% in spleen. Without injury, CD45⁺Col⁺ cells constituted 0.1% in the BM and were undetectable (0.0%, not shown) in spleen. Dot plot is representative of ten independent experiments. c Splenic CD45⁺Col⁺ cells, cultured on plastic in RPMI+10% FCS+TGF-β1 (5 ng/ml), differentiate into α-SMA⁺ myofibroblasts cells. Representative images of three independent experiments are shown

Phenotyping of splenic CD45⁺Col⁺ cells from LPS-treated mice. a Response of splenic CD45⁺Col⁺ cells to LPS. Col-GFP mice are subjected to single or multiple (n=3) LPS injections. The number of CD45⁺Col⁺ cells in the BM and spleen was analyzed 3 or 9 days later, respectively. CD45⁺Col⁺ cells migrated to spleen after single LPS (0.5±0.2%) and multiple LPS injections (4.3±0.9%, of total splenocytes). Mean±SEM are shown (n=13 assays, left panel). Right panel, histograms show MHC II expression by BM and splenic CD45⁺Col⁺ cells, stained with anti-MHC II antibody (blue) or isotype matched control antibody (red). Representative dot plots are shown. b Splenic CD45⁺Col⁺ cells from LPS-treated Col-GFP mice were analyzed by flow cytometry. Representative histograms and dot plots are shown. c Comparison of surface markers expression by BM and splenic CD45⁺Col⁺ cells. CD45⁺Col⁺ cells from BM and spleen of LPS-treated Col-GFP mice are analyzed by flow cytometry for CD11b, F4/80, Gr-1, CD11c, MHC I, MHC II, surface markers, and intracellular cytokines (IFN-γ, TNF-α, and IL-1). Bars represent the mean fluorescent intensity±SEM of three independent experiments. d Electron micrographs of the BM and splenic CD45⁺Col⁺ cells. CD45⁺Col⁺ cells are studied by EM at a magnification of ×6,000, upper panel, and ×10,000, lower panel. Vacuoles are marked with arrows, and electron-dense ER is marked with asterisks. Other features are listed as N (nucleus), MV (microvili), and MPV (micropinocytotic vesicles). EMs are representative images from ten random fields per sample. e BM and splenic CD45⁺Col⁺ exhibited similar morphology, detected by Wright–Giemsa staining, and visualized as round cells, with a basophilic and vacuolated cytoplasm with occasional basophilic granules. Representative images from nine to ten random fields using a ×60 objective

Splenic CD45⁺Col⁺ cells possess anti-microbial properties. a Splenic CD45⁺Col⁺ cells lack phagocytic activity. Splenic MΦ but not splenic CD45⁺Col⁺ fibrocyte-like cells (Sp. F) phagocytosed flouro-ruby dextran (10,000 MW), as detected by flow cytometry after 15 min of co-culturing. Dot plot is representative of three experiments. b Splenic CD45⁺Col⁺ cells release antimicrobial extracellular DNA-traps to entrap and kill L. monocytogenes (Lm). CD45⁺Col⁺ cells co-incubated with L. monocytogenes are stained with anti-mCRAMP Ab, visualized in red (Alexa fluor 568). DNA traps are visualized in blue (Dapi); fibrocytes-like cells are visualized in green (GFP). Bar represents 20 μm. c Release of extracellular traps is associated with death of splenic CD45⁺Col⁺ cells. Splenic CD45⁺Col⁺ cells are co-incubated with L. monocytogenes or PMA for 30 min and analyzed for live/dead cells using Mammalian Cells Viability Kit, right panel. Splenic CD45⁺Col⁺ cells are compared to PMA-treated GFP⁺ B cells (from β-actin-GFP mice). Dead cells and DNA traps are visualized in red (PI). Bar represents 20 μm d Splenic CD45⁺Col⁺ cells reduce viability of L. monocytogenes in vitro after 30 min of co-incubation, as shown in upper and middle panel. Dead bacteria are visualized in red (PI; red arrows) using Live/Dead Bacterial Viability Kit versus live bacteria (green). In addition to fluorescence microscopy, surviving bacteria were quantified by plating surviving colony-forming units on agar plates. Data are shown as mean values±SD of surviving colony-forming units per milliliter. Lower panel, B cells co-cultured with L. monocytogenes serve as control. e Characterization of the extracellular DNA structures formed by splenic CD45⁺Col⁺ cells, untreated or 30 min after incubation with L. monocytogenes, PMA. The number of DNA traps, mCRAMP⁺ cells, viability of trap-forming cells, and dead bacteria is evaluated. B cells, isolated from the same mice and cultured with L. monocytogenes, served as a negative control. The data are presented as percent of positive cells calculated ±SEM, p≤0.005

TGF-β1, LPS, and Listeria monocytogenes induce migration of CD45⁺Col⁺ cells to spleen and liver in vivo. a Col-into-wt mice are i.v. infected with TGF-β1-expressing adenovirus (1×10⁸ pfu) or control adenovirus, or injected with LPS (6 μg/g). After 72 h, GFP⁺ cells (white arrows) are detected by immunofluorescence (GC-germinal center, RP-red pulp) or flow cytometry (mean fluorescent intensity±SEM (n=5 mice/group, *p<0.001). Collagen deposition was estimated by TriChrome staining (blue arrows), which shows lack of collagen deposition in spleen. b Mobilization of CD45⁺Col⁺ cells in response to L. monocytogenes infection. Col-into-wt mice are infected with L. monocytogenes (4×10⁶ cfu) for 24 h. GFP⁺ cells are detected in livers and spleens but not kidneys, as visualized by fluorescent microscopy using a ×10 objective, left panel. The bacterial load of organ homogenates is calculated for liver (8.6±2×10⁷ cfu/g), spleen (3.7±2×10⁸ cfu/g), and kidney (8±4×10⁵ cfu/g). The data represent the average of three mice. c The effect of splenic CD45⁺Col⁺ cells on liver fibrosis is studied in splenectomized mice. Outline of the experiment: spleen is surgically removed (−Sp) in experimental group of mice. Left panel, fluorescent micrographs of liver obtained from splenectomized or sham-operated CCl₄-treated Col-into-wt mice and Col-GFP mice. Phase contrast micrographs are TriChrome staining of liver tissues, obtained for each group of mice. Right panel, liver tissues from splenectomized or sham-operated Col-into-wt mice are analyzed for GFP⁺ or TriChrome-positive areas. mRNA levels of collagen α1(I) expression in splenic CD45⁺Col⁺ cells was compared with hepatic stellate cells (SC), as detected by RT-PCR. The data are presented as mean±SEM, *p<0.001

Comparison of splenic CD45⁺Col⁺ cells and macrophages. a Gene expression microarray of splenic CD45⁺Col⁺ cells. Splenic CD45⁺Col⁺ cells from LPS-treated mice and peritoneal macrophages (MΦ) from thioglycollate-stimulated mice, unstimulated (quiescent), or Kdo₂-lipid A-treated (100 ng/ml, 6 h, activated) are compared by gene expression microarray. Venn diagram shows the number of shared and distinct genes. b Gene expression overlap identified for each group, presented as GO TERM with the p values indicated for each group. c Gene expression analysis of fibrocyte precursor-specific genes by RT-PCR. Relative mRNA levels are calculated for qMΦ, aMΦ, and splenic FPs after normalization to GAPDH gene using the ΔΔ CT method. Genes are listed as Chi3l3 (chitinase 3-like 3), Ngp (neutrophilic granular protein), IL-18R (interleukin 18 receptor), mCRAMP (cathelicidin antimicrobial peptide), and Vcam-1 (vascular cell adhesion molecule). Fold induction of gene expression in splenic CD45⁺Col⁺ cells is shown in comparison with the highest value detected in qMΦ or aMΦ, p>0.0001

Alternative functions of splenic CD45⁺Col⁺ fibrocyte-like cells. a Splenic CD45⁺Col⁺ fibrocyte-like cells possess properties of antigen presenting (APC) cells. Splenic fibrocyte-like cells present antigens on MHC I (H2-K^b) and MHCII (I-Ab) in vitro, left panel. DCs and splenic CD45⁺Col⁺ cells (5×10⁴ cells) untreated or loaded with OVA^257–264 or OVA^323–339 are co-cultured for 4 days with CFSE-labeled CD8⁺ OT-I or CD4⁺ OT-II T cells (1.5×10⁵), respectively. Proliferation is measured by CFSE dilution in activated CD44⁺ T cells. Representative dot plots are shown. Splenic CD45⁺Col⁺ cells induce proliferation of adoptively transferred CFSE-OT-I/bm1 CD8⁺ T cells in Act-mOVA/bm1 mice, right panel. Proliferation of CFSE-labeled T cells in the liver, spleen, and peripheral lymph nodes (LN) was analyzed 4 days later by flow cytometry. Data represent mean±SEM. b A subset of splenic CD45⁺Col⁺ cells co-express MHC II and mCRAMP. Purified from spleens LPS-treated mice, CD45⁺Col⁺ cells are co-stained with anti-mCRAMP and anti-MHC II antibodies. Upper panel, co-localization of MHC II (shown in white) and mCRAMP (red) is detected in 43±7% of cells. Bar represents 30 μm. Lower panel, representative images are magnified five times; double positive cells are marked with arrows c Splenic CD45⁺Col⁺ cells differentiate into myeloid (CD11b⁺CD14⁺F4/80⁺) or dendritic (CD11b⁺CD11c⁺) cells in response to M-CSF (30% L-cell medium) or GM-CSF (20 ng/ml), respectively, detected by flow cytometry, upper panel. Lower panel, phagocytosis of FITC-labeled Escherichia coli by M-CSF-differentiated splenic CD45⁺Col⁺ cells or BM-derived macrophages. The number of phagocytosed E. coli-FITC particles increases over time. Representative images of three independent experiments are shown d Splenic fibrocytes give rise to CD11b⁺ (76%), CD11b⁺CD11c⁺ (2.6%), CD11b⁻CD11c⁺ (3.7%), and Gr-1⁺ (9%) cells, when transferred into sublethally irradiated (600 Rad) CD45.1⁺ mice (n=10). Fibrocyte differentiation is accompanied by the loss of collagen-GFP expression. Dot blot analysis: representative images of five experiments are shown