Elevated frequencies of programmed death ligand 2 (PDL2) in macrophages of BALB/c mice model of liver fibrosis associated with decreased CD80 counts
Polarizing of monocytes into M1 and M2 macrophages are an important step in liver fibrosis progression and regression . We characterized their role during fibrogenesis. Hepatic fibrosis was induced in BALB/c mice by biweekly IP CCl4 injections for 4 weeks and was compared with naive vehicle-treated mice.Liver macrophages were isolated as described in materials and methods along week 1 to week 4 of CCl4 injections. Macrophages were then stained for M1 and M2 subpopulations for CD80 and anti-CD273, respectively. iNOS for M1 macrophages and anti-CD206 for M2 macrophages were also used as an additional marker to profile macrophages subpopulations. Fig. 1A shows a represented dot plot of flow cytometry analysis of the isolated polymorphonulcear cells (Gate 1; PMN following Ficoll separations according to their size (FSC-H) and granularity (SSC-H). Fig. 1B shows the whole population stained with the pan-leukocytes marker anti-CD45-FITC. A gate (Gate 2) was drawn on our potential macrophages populations identified as CD45-positive cells with high SSC-A. For further identifying our macrophages, Fig. 1C demonstrates plotted population stained with both anti-CD68-APC (pan-macrophages marker) and a gate (Gate 3) was set on CD68-poistive population. We also confirmed pan-macrophages populations with staining with F480-pacrific blue and similar results were obtained (data not shown) Fig. 1D shows a representative distribution of macrophages subpopulations of M1 and M2 identified as CD80-PE and CD273-PerCP, respectively. Of the total evaluated CD45+ /CD68+ cells, averages of around 13.3% of CD80 and 12.4% of iNOS M1-macrophages were expressed in naïve mice. These percentages were decreased in early fibrosis (w1 and w2 of CCl4) and ended in advanced fibrosis of w4 to 1.97% and 5.1%, respectively (Fig. 1E; P<0.05). In contrast, expressions of CD273 and CD206 on M2-macrophages of naïve mice were 19.8% and 35.6%, respectively. Both CD273 and CD206 expressions showed to be elevated significantly through fibrosis severities to 27.9% and 48%, respectively (p<0.05). There were no significance differences in CD273 and CD206 expressions between week 1 along week 4 of fibrosis (Fig. 1F).The overall M1 to M2 ratio showed a decreased in M1/M2 percentages (Fig. 1G), indicating an alterations in macrophages subpopulations counts where M2 were the dominant populations and increased in fibrosis while M1 showed decreased in their counts.
M2-macrophages phenotype alterations accompanied by decrease in IL–12, VDR expressions and increased in their TGF- productions
In order to evaluate whether changes in macrophages count were accompanied with phenotypic alterations; we have determined M2-macrophages release of interferon- (INF-), transforming growth factor - (TGF- and Interleukin 12 (IL–12).
IL–12 is produced by activated antigen-presenting cells (dendritic cells, macrophages) . It promotes the development of Th1 responses and is a powerful inducer of IFNγ production by T and NK cells . TGF is a powerful modulator of inflammation, and has been implicated in the regulation of leukocyte and vascular endothelial cell activation . Mice lacking the TGF gene die of widespread inflammatory disease either in utero or shortly after birth [21, 22].
Fig. 2A shows flow cytometry analysis of the percentages of INF- significantly decreased from 65.4±11% in naïve mice to 55.4±9% (w1, p = 0.04), 51.9±7% (w2, p = 0.03), 45.8±5% (w3, p = 0.03), and 36.5±5% (w4, p = 0.001). In parallel, Fig. 2B shows IL12 percentages were also gradually decreased from 13.4±3.9% in naïve mice to 10.375±2.7% (w1, p = 0.04), 4.025±1.6% (w2, p = 0.03), 3.7±0.3% (w3, p = 0.03), and 1.075±0.1% (w4, p = 0.001). In contrast to these data, TGF- expressions were gradually elevated in the M2-macrophages from 19.5±3.7% in naïve mice to 29.0±5.1%, 35.1±7.9%, 41.3±7%, and 51.5±9% in w1, w2, w3 and w4, respectively (Fig. 2C, p<0.05 between all groups). M2-macrophages expressions of VDR were 55.9±8.1% in naïve mice and were gradually decreased to 49±8%, 40±6%, 37±5.8% and 29±4.1% in w1, w2, w3 and w4, respectively (Fig. 2D, p<0.05 between all groups). These results suggest M2-macrophages lost their inflammatory response exhibiting INF- during fibrosis progressions (reduce INF-), probably due to decrease in their IL–12 secretions, and in contrast they had an anti-inflammatory response through their release to TGF-Decrease in VDR expression on M2-macrophages during fibrosis progressions could suggest its correlation with their pro-fibrogenic properties
Interleukin–4 ameliorated metabolic markers of insulin, vitamin D and vitamin D receptor associated with CCl4 fibrosis model
We sought to further explore changes in phenotype of the dominant M2-macrophages populations; therefore, we specified metabolic markers for their correlation to macrophages activity through inductions of interleukin–4 (IL–4) in an acute and chronic model of CCl4. IL–4 showed to restore insulin sensitivity  and in other studies speeds up fibrosis reversal . In our current study, we evaluated serum insulin levels, vitamin D as well as vitamin D receptor. Results from preclinical studies showed that vitamin D (or calcitriol) administration reduced the levels of blood glucose and improved insulin sensitivity in diabetic mice  and attenuated the fibrosis .
Fig.3 shows effects of IL–4 on serum insulin, vitamin D and vitamin D receptor levels in acute injections of CCl4 (2 weeks) as well in chronic injections of CCl4 (4 weeks). Fig. 3A shows no significant changes in serum insulin levels in the acute phase of the disease as compared to mice with no CCl4 treatments while the chronic phase of liver fibrosis exhibit low insulin levels. While inductions of i.p injections of IL–4 did not affect insulin levels in acute phase, IL–4 inhibited decrease in insulin levels in the chronic model of CCl4 (P = 0.01) and maintained serum insulin comparable to untreated mice. Similar data were obtained for serum vitamin D levels illustrated in Fig. 3B. In Fig. 3C, serum VDR showed to decrease in acute CCl4 injections from 730.35 in untreated groups to 376.25 in acute CCl4 injection with a further decrease to 282 ng/ml in chronic injections. IL–4 inductions while inhibited decrease in VDR in acute model it elevated serum VDR in chronic model to levels exceeding untreated group (P = 0.0012). IL–4 inductions in CCl4-untreated mice had similar patterns to naïve animals in all metabolic profile (data are not shown). The results emphasize the association between fibrosis progressions and alteration in metabolic profile such as insulin resistance. IL–4 is suggested to activate insulin receptor and promote signaling pathway related to TGF- production and chemotaxis necessary for extracellular matrix synthesis. In addition, our data refer to VDR as an early metabolic marker to predict fibrosis progressions or regressions.
Fibrotic BALB/c mice develop severe fibrosis and inflammation with increased intra-hepatic macrophages while reversed by IL–4
In order to evaluate whether our metabolic data together with the effects of IL–4 are correlated with alteration in fibrosis progressions; we have performed fibrosis marker of the αSMA expressions, ALT levels and histological liver assessments.
H&E immunohistochemical staining (Fig. 4A) for the animal groups showed lymphocytes infiltration in the fibrotic mice that were attenuated with IL–4 inductions. No inflammatory infiltrates were seen in H&E staining of naïve untreated. Fig. 4B shows the lack of fibrosis stained by Sirius-Red in naïve untreated mice. CCl4 induction showed increased formation of red fibrosis septae in CCl4 and less prominent in IL–4 induce fibrotic mice. To define infiltrate of macrophages in these livers; F4/80 stain for the macrophage marker was assessed. Fig.4C shows cell infiltrate including macrophages (F4/80-positive cells) in the fibrotic animals. Fibrotic mice with IL–4 showed similar staining to untreated mice with less macrophage infiltrates.
Fig. 4D shows expressions of αSMA-mRNA in livers with and without fibrosis with IL–4 inductions. CCl4-induction provoked increased fibrosis and αSMA expression was significantly increased 4.2±0.5 folds in the CCl4 animals as compared to the untreated counterparts (P = 0.018). Fibrotic animals with IL–4 had reduced expressions of αSMA mice as compared to animals with the CCl4 alone (P = 0.03) and had similar expressions to untreated mice (P = ns). These results confirmed same pattern obtained from histology assessments. There were no differences in the CYP2e activity of liver extracts from two fibrotic groups (data not shown). Serum ALT levels (Fig. 4E) were significantly (P<0.05) increased from 57.3±18.7 in naïve mice to 228±29, 404.5±89, 333.3±77 and 178±51 IU/L in CCl4- fibrotic mice of week 1, 2, 3 and 4, respectively. On the other hand, animals with 2 weeks of CCl4 were induced with IL–4 had serum ALT levels reduced following 1 week of induction with prominent reductions in ALT levels following 2 weeks (p = 0.01). The ALT results were in line with obtained H&E staining.
Serum obtained from fibrotic mice following IL–4 inductions showed increased levels of TGF- (Fig. 4 F; p = 0.031), elevated levels of INF- (Fig. 4 G; p = 0.002) as well IL–12 (Fig. 4 H; p = 0.014) as compared to fibrotic mice with no inductions. These results indicate IL–4 ability to accelerate liver fibrosis (through TGF-) and therefore inhibit hepatic stellate cells activations (decreased in SMA), minimize liver injury through differentiation of macrophages by IL–12 and promote INF- expressions and as a consequent enhance their activity. Taking all together, IL–4 showed to enhance liver inflammation and fibrosis progression thorough ameliorating vitamin D, VDR and insulin suggesting IL–4 effects as speeds up fibrosis reversal.