*3.4. Fibrocyte Ablation Lead to A Reduction of Hepatic IL-1*β *Levels*

Next, we evaluated the hepatic infiltration and proliferation of inflammatory cells. While histological grading, performed on routine hematoxylin/eosin-staining, did not retrieve significant differences in result of fibrocyte-ablation (Table S6), immunohistochemical staining and subsequent morphometric analysis of the pan-leucocyte marker CD45 hinted at a decreased number of CD45-positive cells in the liver (*p* = 0.054; Figure 4a,b).

**Figure 4.** Fibrocyte ablation lead to a reduction of hepatic IL-1βlevels. (**a**) Immunohistochemical staining of CD45 (grey) and (**b**) subsequent morphometric analysis revealed a tendentially reduced number of leukocytes in the liver of fibrocyte-ablated mice. Magnification 200×, bar 50μm. Mann-Whitney *U* test was applied. (**c**) Multiplex ELISA demonstrated a reduction of IL-1β protein levels while none of the other cytokines were significantly regulated. Absolute concentrations and individual *p*-values are provided in Figure S8. (**d**) qRT-PCR showed no regulations in a panel of inflammatory genes (full data in Figure S5d–i). (**e**–**g**) In comparison to healthy supercontrols (*n* = 8, dotted bars), absolute quantification of hepatic eicosanoids revealed a notable decrease of all but one analyte (5,6-EET + DHET) in consequence of the TAA-treatment. The level of LTB4 is considerably lower in FC-ablated mice (*n* = 15, blue), compared to controls (*n* = 15, black). Mean of three measurements + SEM are depicted.

In order to assess paracrine inflammatory functions of fibrocytes, we evaluated various cytokines on a transcriptional and protein level. Proteome Profiling of 111 cytokines was performed, yet none of the suspected mediators were strongly regulated (Figure S7). Multiplex ELISA-data of common inflammatory cytokines (Figure 4c, full data in Figure S8) revealed that the level of IL-1β was significantly decreased in fibrocyte-ablated mice (13.7 ± 0.98 vs. 12.9 ± 1.12 ng/g liver; *p* = 0.044). Moreover, genes encoding inflammatory markers (*Tnf*, *Ccl3*, and *Ccl12*) were among the strongest regulated analytes in the gene expression array (Figure S3). Significantly regulated- and further genes of interest were therefore assessed using quantitative real-time PCR (Figure 4d), yielding no significant regulations.

Since FC are known to express cysteinyl leukotrienes (CysLTs), we also sought to determine the hepatic levels of several eicosanoids (Figure 4e–g). While CysLTs were not detectable in our experimental setup, other changes in the eicosanoid profile occurred in response to the fibrogenic stimulus. The concentration of leukotriene B4 (LTB4), several isomers of epoxyeicosatrienoic acid (EETs), 15-deoxy-delta-12,14-prostaglandin J2 (15-d.-PGJ2), and prostaglandin E2 (PGE2) decreased in TAA-treated groups. FC ablation lead to a subtle attenuation of the EETs and a notable decrease of LTB4 (Figure 4e).
