Acute Liver Injury after CCl4 Administration Is Independent of Smad7 Expression in Myeloid Cells

Myeloid cells are essential for the initiation and termination of innate and adaptive immunity that create homeostasis in the liver. Smad7 is an inhibitor of the transforming growth factor β (TGF-β) signaling pathway, which regulates inflammatory cellular processes. Knockdown of Smad7 in hepatocytes has been shown to promote liver fibrosis, but little is known about the effects of Smad7 in myeloid cells during inflammatory responses in the liver. Using mice with a myeloid-specific knockdown of Smad7 (LysM-Cre Smad7fl/fl), we investigated the impact of Smad7 deficiency in myeloid cells on liver inflammation and regeneration using the well-established model of CCl4-mediated liver injury. Early (24/48 h) and late (7 d) time points were analyzed. We found that CCl4 induces severe liver injury, with elevated serum ALT levels, centrilobular and periportal necrosis, infiltrating myeloid cells and an increase of inflammatory cytokines in the liver. Furthermore, as expected, inflammation peaked at 24 h and subsided after 7 d. However, the knockdown of Smad7 in myeloid cells did not affect any of the investigated parameters in the CCl4-treated animals. In summary, our results suggest that the inhibition of TGF-β signaling via Smad7 expression in myeloid cells is dispensable for the induction and control of acute CCl4-induced liver injury.


Introduction
The response of the liver to injury, irrespectively of aetiology, involves different cell types such as hepatocytes, hepatic stellate cells (HSCs) and macrophages (Kupffer cells (KCs)), and encompasses different phases.Characterized by cellular stress leading to various kinds of hepatocyte death, this decline of hepatocytes activates a cascade of signals that initiates inflammation and tissue repair mechanisms.After liver injury, complete repair and regeneration are possible, but if the injury becomes chronic it can also lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC).
Hepatocyte damage leading to fibrosis can be induced via oxidative stress that induces activation of HSCs and Kupffer cells in which TGF-β is a key mediator [1].Signaling via the TβRI leads to the recruitment of receptor-activated Smads (R-Smads), including Smad2 and Smad3, which in turn complex with the common Smad (C-Smad) Smad4.This complex then translocates into the nucleus where it regulates transcription of TGF-β target genes.TGF-β mediated signals can be regulated in several ways.Ubiquitinylation of phosphorylated Smad molecules in the nucleus leads to their proteasomal degradation by which the level of activated Smad proteins is controlled.Furthermore, TGF-β induced target genes include the inhibitory Smad (I-Smads) molecules 6 and 7, of which Smad7 can counteract TGF-β signaling via its association with the TβRI.The interaction of Smad7 with the TβRI can, on the one hand, inhibit the recruitment and activation of the R-Smads.On the other hand, it can lead to TβRI receptor degradation via the recruitment of the ubiquitin E3 ligases Smurf1 and 2 [2].TGF-β-dependent signals are important regulators of cell proliferation and survival.TGF-β-induced cytostasis functions via the repression of growth-promoting transcription factors (e.g., c-Myc, Id1-3) and the induction of specific CDK inhibitors [3].
Upon liver injury, several hepatic cell types, such as organ-resident macrophages (i.e., Kupffer cells) and hepatic stellate cells (HSCs), readily produce TGF-β.This is pivotal for fibrosis progression, as TGF-β promotes HSC activation and transdifferentiation into myofibroblasts that are then stimulated to proliferate according to further TGF-β signals [1].Both depletion of hepatic stellate cells [4] and depletion of macrophages [5] results in the attenuation of liver fibrosis, which is characterized by a marked decrease in matrix deposition and a reduction of HSC activation.Moreover, in several liver injury models, interference with pro-fibrotic TGF-β signaling attenuates the severity of disease or injury.Direct inhibition of TGF-β receptor signaling in hepatocytes promotes liver regeneration after acute CCl 4 intoxication [6].Expression of the negative regulator of TGF-β signaling Smad7 leads to diminished sensitivity to TGF-β signals.For instance, deletion of Smad7 in hepatocytes aggravates liver injury and steatosis after alcohol feeding [7] as well as fibrogenesis in an acute CCl 4 -mediated injury model [8], and it accelerates chemically induced HCC development [9].Contrary, hepatic overexpression of Smad7 inhibits fibrogenesis in CCl 4 -mediated injury [10].
In liver injury, macrophages produce TGF-β and are important for cross talk with HSCs, leading to the activation and generation of myofibroblasts that produce extracellular matrix components for fibrosis.It is, however, unclear how TGF-β signaling and Smad7-mediated counter regulation of TGF-β signaling is regulated in hepatic macrophages during liver injury and inflammation.Overexpression of Smad7 in intestinal macrophages is associated with their pro-inflammatory activation [11], and shRNA-mediated knockdown of Smad7 is currently being tested as a therapy for inflammatory bowel disease [12].Here, we investigated the contribution of myeloid-expressed Smad7 on disease severity in an acute model of liver injury and regeneration after a single application of CCl 4 using a myeloid-specific Smad7 knockdown mouse model (LysM-Cre Smad7 fl/fl ).Our results show that a single application of CCl 4 in LysM-Cre Smad7 fl/fl knockdown and their Smad7 fl/fl littermate controls led to liver injury with elevated liver enzymes in the serum, areas of necrosis in histological analyses and infiltration of immune cells, as well as the induction of liver regeneration.However, the knockdown of Smad7 in LysM-expressing cells did not influence the severity of any of these parameters that are indicative of liver injury and regeneration.Thus, negative regulation of TGF-β signaling via Smad7 expression in myeloid cells does not contribute to CCl 4 -mediated liver injury and regeneration.

Liver Injury after CCl 4
Administration is Similar in Myeloid-Specific Smad7 Knockdown and Wild-Type Controls TGF-β signaling is pivotal in liver injuries leading to fibrosis [13], and expression of the negative regulator of TGF-β signaling, Smad7, can modulate liver injury, fibrosis and also cancer development in (for instance) hepatocytes [7,9,10].Hepatic macrophages produce high amounts of TGF-β and are pivotal for fibrosis progression via the activation of HSCs [1].In order to investigate whether Smad7-dependent modulation of TGF-β signaling influences the severity of liver injury in macrophages, we generated a myeloid-specific Smad7 knockdown mouse model via backcrossing a LysM-Cre mouse line with a floxed Smad7 mouse line, in which the Cre-mediated recombination results in the deletion of exon 1 from the Smad7 gene [14].As hepatocyte-specific deletion of Smad7 has been described as leading to spontaneous liver dysfunction [7], and TGF-β signaling is in general important for the development of various tissues, we first analyzed whether Smad7 knockdown in LysM-Cre Smad7 fl/fl mice was efficient.Smad7 mRNA was significantly reduced in splenic CD11b + cells and bone-marrow-derived macrophages (BMDMs) from LysM-Cre Smad7 fl/fl animals, whereas Smad7 mRNA was readily detected in both splenic CD11b + cells and BMDMs from Smad7 fl/fl control animals and in splenic CD8 + T cells from both LysM-Cre Smad7 fl/fl and Smad7 fl/fl control animals.This indicates that the LysM-Cre-mediated deletion of Smad7 is both highly efficient as well as specific (Figure 1A).leading to spontaneous liver dysfunction [7], and TGF-β signaling is in general important for the development of various tissues, we first analyzed whether Smad7 knockdown in LysM-Cre Smad7 fl/fl mice was efficient.Smad7 mRNA was significantly reduced in splenic CD11b + cells and bone-marrowderived macrophages (BMDMs) from LysM-Cre Smad7 fl/fl animals, whereas Smad7 mRNA was readily detected in both splenic CD11b + cells and BMDMs from Smad7 fl/fl control animals and in splenic CD8 + T cells from both LysM-Cre Smad7 fl/fl and Smad7 fl/fl control animals.This indicates that the LysM-Cre-mediated deletion of Smad7 is both highly efficient as well as specific (Figure 1A).We injected mice with a single dose of CCl4 i.p. and analyzed various parameters at 24 and 48 h after the application.Exposure to CCl4 leads to centrilobular hepatic necrosis, due to the generation We injected mice with a single dose of CCl 4 i.p. and analyzed various parameters at 24 and 48 h after the application.Exposure to CCl 4 leads to centrilobular hepatic necrosis, due to the generation of highly toxic metabolites via cytochrome P450 2E1 [15].The subsequent hepatocyte death leads to the release of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).Mice receiving CCl 4 had markedly increased ALT and AST levels in the serum 24 and 48 h after injection, indicating hepatocyte loss, although their body weight did not change (Figure 1B-D).H & E staining revealed extensive liver injury after CCl 4 administration (Figure 1E), but detailed analysis could not reveal an impact of Smad7 deficiency on myeloid cells for parameters such as area of necrosis (Figure 1F) or infiltrating immune cells, such as neutrophils, eosinophils or lymphocytes (Figure 1G).In summary, these data indicate that Smad7 expression in myeloid cells does not influence the extent of liver injury nor the composition of infiltrating immune cells after CCl 4 -induced liver damage.

Neither Immune Cell Infiltration nor Inflammatory Gene Expression is Affected by the Loss of Smad7 Expression in Myeloid Cells after CCl 4 Administration
We next evaluated in more detail the myeloid cell infiltration into injured livers after CCl 4 injection.Using histological staining we analyzed the amount of infiltrating myeloperoxidase (MPO)-expressing cells and F4/80-expressing cells into the livers of CCl 4 -treated animals.Both MPO (Figure 2A)-and F4/80 (Figure 2B)-positive cells were increased in the liver after CCl 4 treatment, but myeloid Smad7 deficiency did not alter their proportion (Figure 2C).To be able to differentiate in more detail between the myeloid cells in the livers of CCl 4 -treated LysM-Cre Smad7 fl/fl and Smad7 fl/fl animals, we isolated the non-parenchymal cells (NPC) from these livers and stained the cells for CD11b, CD11c, SiglecF, Ly6G and Ly6C.Flow cytometric analyses revealed a large increase in total CD11b-positive cells in CCl 4 -treated animals compared to the non-treated controls (Figure 2D).After 24 h, the majority of myeloid cells were CD11b + CD11c -SiglecF -Ly6G + neutrophils, whereas after 48 h most CD11b + cells were CD11c -SiglecF + and CD11c -SiglecF -Ly6G -Ly6C + , indicating that in the course of the inflammatory reaction due to CCl 4 injection, neutrophils, eosinophils and inflammatory monocytes are recruited into the liver (the gating strategy in Figure S1).However, the knockdown of Smad7 in myeloid cells did not affect the recruitment of any of the investigated myeloid subsets (Figure 2E).Although the number of infiltrating myeloid cells may not be affected, their functionality may be affected.In order to elucidate whether loss of Smad in myeloid cells can affect their function, we analyzed inflammatory gene expression in the livers of CCl 4 -treated LysM-Cre Smad7 fl/fl and Smad7 fl/fl animals via quantitative reverse transcriptase PCR (qPCR) (Figure 3).Expression of mRNA of the proinflammatory genes Il1b, Il6, Tnf and Ccl2 was highly induced early after CCl 4 .Expression of anti-inflammatory Il10 mRNA also increased early after CCl 4 administration.However, we did not observe any effects of Smad7 knockdown in myeloid cells here either.All investigated parameters were similarly regulated in LysM-Cre Smad7 fl/fl and Smad7 fl/fl animals.Thus, the infiltration of inflammatory myeloid cells and cytokine/chemokine gene expression was not regulated by Smad7 expression in myeloid cells after CCl 4 application in vivo.

Smad7 Expressing Myeloid Cells do not Affect the Regenerative Capacity of the Liver after CCl4 Mediated Injury.
Thus far we found no evidence of the involvement of myeloid-expressed Smad7 in the early phases of the immune response after CCl4 administration in mice.In addition to the profound and rapid inflammatory response in the liver, administration of CCl4 also leads to a well-defined regenerative response [16] that is characterized by the induction of cell-cycle proteins and hepatocyte proliferation during the first seven days after CCl4 application.To analyze the regenerative response, we analyzed the expression of several cell-cycle proteins (a.o.cyclins and cyclin-dependent kinases)

Smad7 Expressing Myeloid Cells Do not Affect the Regenerative Capacity of the Liver after CCl 4 Mediated Injury
Thus far we found no evidence of the involvement of myeloid-expressed Smad7 in the early phases of the immune response after CCl 4 administration in mice.In addition to the profound and rapid inflammatory response in the liver, administration of CCl 4 also leads to a well-defined regenerative response [16] that is characterized by the induction of cell-cycle proteins and hepatocyte proliferation during the first seven days after CCl 4 application.To analyze the regenerative response, we analyzed the expression of several cell-cycle proteins (a.o.cyclins and cyclin-dependent kinases) that are induced to facilitate hepatocyte proliferation and liver regeneration.We could observe early induction of a.o.Ccnd1, Cdk4, Cdk2, Cdc25a and Cdkn1a mRNA, which was downregulated over the seven-day analysis period.mRNA expression of other cell-cycle proteins such as Cdk1 and Ccnb1 reached peak values at 48 h and declined after seven days.However, we could not observe changes in expression levels of these genes due to Smad7 knockdown in LysM-expressing myeloid cells.Corresponding to the mRNA expression analyses of the cell-cycle genes, we found higher levels of Ki67-positive hepatocytes after CCl 4 injection in histology (Figure 4B), but also here we found no evidence that myeloid-specific Smad7 deficiency affected this response after scoring and counting Ki67 positive hepatocytes (Figure 4C).Together, these data show that Smad7 expression in myeloid cells does not play a role in the liver regenerative response after CCl 4 -induced liver injury.
Int. J. Mol.Sci.2019, 20, x FOR PEER REVIEW 7 of 11 that are induced to facilitate hepatocyte proliferation and liver regeneration.We could observe early induction of a.o.Ccnd1, Cdk4, Cdk2, Cdc25a and Cdkn1a mRNA, which was downregulated over the seven-day analysis period.mRNA expression of other cell-cycle proteins such as Cdk1 and Ccnb1 reached peak values at 48 h and declined after seven days.However, we could not observe changes in expression levels of these genes due to Smad7 knockdown in LysM-expressing myeloid cells.
Corresponding to the mRNA expression analyses of the cell-cycle genes, we found higher levels of Ki67-positive hepatocytes after CCl4 injection in histology (Figure 4B), but also here we found no evidence that myeloid-specific Smad7 deficiency affected this response after scoring and counting Ki67 positive hepatocytes (Figure 4C).Together, these data show that Smad7 expression in myeloid cells does not play a role in the liver regenerative response after CCl4-induced liver injury.cell-cycle gene mRNA.Histology of liver tissue was performed on formalin-fixed paraffin-embedded liver samples.

mRNA Isolation and Quantitative RT-PCR
Murine liver tissue was flash frozen in liquid nitrogen and stored at −80 • C until further processing.mRNA was isolated using a RNeasy Mini kit from Qiagen (#74104, Hilden, Germany) following the manufacturer's instruction.gDNA was digested separately using the DNase Treatment & Removal kit (AM1906, Invitrogen, ThermoFisher Scientific, Bremen, Germany) from Invitrogen. mRNA was transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit from Applied Biosystems (#4368813).Quantitative RT-PCR (qPCR) was either performed with exon spanning primers for relevant inflammatory and cell-cycle genes using the PowerUp SYBR Green Master Mix (ThermoFisher Scientific, Bremen, Germany) from Applied Biosystems (A25742), or with Taqman gene expression assays (Applied Biosystems, ThermoFisher Scientific, Bremen, Germany) for muSmad7 and the reference genes muGapdh/muHprt1/hu18S on a Viia QuantStudio 7 (ThermoFisher Scientific, Bremen, Germany) (primers used are listed in Table S1).Relative mRNA expression levels were calculated using the ∆Ct method.

Figure 1 .
Figure 1.Liver enzymes and histological damage parameters are not changed due to myeloid deletion of Smad7 after CCl4 administration.(A) Smad7 mRNA expression by quantitative real time PCR on splenic CD11b+, CD8+ and bone-marrow-derived macrophages (BMDMs) from Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates (n = 4).(B-G) The 8-to 10-week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl4 in corn oil and sacrificed at the indicated time points.(B) Serum ALT, (C) serum AST, and (D) body weight were determined 24 and 48 h after i.p. injection of CCl4.(E) Representative H&E staining of Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates.Representative pictures are shown at 20x magnification.(F) Histopathological score of centrilobular and periportal necrosis in H&E sections.(G) Histopathological score of lobular neutrophil, lymphocyte and eosinophil infiltration in H&E sections.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate the mean ± SEM. * p ≤ 0.05 calculated by Student's t-test, n.s.= not significant.No other significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in the data from panels B-D, F and G.

Figure 1 .
Figure 1.Liver enzymes and histological damage parameters are not changed due to myeloid deletion of Smad7 after CCl 4 administration.(A) Smad7 mRNA expression by quantitative real time PCR on splenic CD11b+, CD8+ and bone-marrow-derived macrophages (BMDMs) from Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates (n = 4).(B-G) The 8-to 10-week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl 4 in corn oil and sacrificed at the indicated time points.(B) Serum ALT, (C) serum AST, and (D) body weight were determined 24 and 48 h after i.p. injection of CCl 4 .(E) Representative H & E staining of Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates.Representative pictures are shown at 20x magnification.(F) Histopathological score of centrilobular and periportal necrosis in H & E sections.(G) Histopathological score of lobular neutrophil, lymphocyte and eosinophil infiltration in H & E sections.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate the mean ± SEM. * p ≤ 0.05 calculated by Student's t-test, n.s.= not significant.No other significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in the data from panels (B-D,F,G).

Figure 2 .
Figure 2. Myeloid cell infiltration after CCl4 administration in LysM-Cre Smad7 fl/fl mice.The 8-to 10week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl4 in corn oil or left untreated and sacrificed at the indicated time points.(A,B) Immunohistochemical staining for myeloperoxidase (MPO) and F4/80 in liver sections of mice injected with CCl4.Representative pictures are shown at 40x magnification.(C) Graphs show enumeration of MPO-and F4/80-positive cells per mm 2 .(D,E) Non-parenchymal cells (NPC) were isolated and stained for CD11b, CD11c, SiglecF, Ly6G and Ly6C.Staining with a fixable live/dead stain identified living cells.(D) Representative dot plots showing percentages (numbers in black rectangles) of viable CD11b + cells in livers from CCl4-treated and non-treated mice.(E) Percentages of CD11b + cells within live cells and percentages of SiglecF + , Ly6G + or Ly6C + cells within CD11b + cells in CCl4-treated Smad7 fl/fl and LysM-Cre Smad7 fl/fl mice after 24 and 48 h.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate the mean ± SEM.Statistical significance was calculated by ANOVA, but no significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in the data from panels (C) and (E).

Figure 2 .
Figure 2. Myeloid cell infiltration after CCl 4 administration in LysM-Cre Smad7 fl/fl mice.The 8-to 10-week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl 4 in corn oil or left untreated and sacrificed at the indicated time points.(A,B) Immunohistochemical staining for myeloperoxidase (MPO) and F4/80 in liver sections of mice injected with CCl 4 .Representative pictures are shown at 40x magnification.(C) Graphs show enumeration of MPO-and F4/80-positive cells per mm 2 .(D,E) Non-parenchymal cells (NPC) were isolated and stained for CD11b, CD11c, SiglecF, Ly6G and Ly6C.Staining with a fixable live/dead stain identified living cells.(D) Representative dot plots showing percentages (numbers in black rectangles) of viable CD11b + cells in livers from CCl 4 -treated and non-treated mice.(E) Percentages of CD11b + cells within live cells and percentages of SiglecF + , Ly6G + or Ly6C + cells within CD11b + cells in CCl 4 -treated Smad7 fl/fl and LysM-Cre Smad7 fl/fl mice after 24 and 48 h.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate the mean ± SEM.Statistical significance was calculated by ANOVA, but no significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in the data from panels (C,E).

Figure 3 .
Figure 3. Inflammatory gene expression in LysM-Cre Smad7 fl/fl after CCl4 injection.The 8-to 10-weekold Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl4 in corn oil and sacrificed at the indicated time points.As a control, mice were left untreated.At the indicated times, liver samples were taken for quantitative real-time PCR for pro-(Il1b, Tnf, Ifng, Il6, Ccl2) and antiinflammatory (Il10) genes.Relative mRNA expression levels of target genes are depicted calculated in relation to the expression of the reference gene mRNA by the Ct method.The dotted line indicates expression in non-treated control animals.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate mean ± SEM.Statistical significance was calculated by ANOVA, but no significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in these data.

Figure 3 .
Figure 3. Inflammatory gene expression in LysM-Cre Smad7 fl/fl after CCl 4 injection.The 8-to 10-week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl 4 in corn oil and sacrificed at the indicated time points.As a control, mice were left untreated.At the indicated times, liver samples were taken for quantitative real-time PCR for pro-(Il1b, Tnf, Ifng, Il6, Ccl2) and anti-inflammatory (Il10) genes.Relative mRNA expression levels of target genes are depicted calculated in relation to the expression of the reference gene mRNA by the ∆Ct method.The dotted line indicates expression in non-treated control animals.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate mean ± SEM.Statistical significance was calculated by ANOVA, but no significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in these data.

Figure 4 .
Figure 4. Cell-cycle gene expression in LysM-Cre Smad7 fl/fl animals after CCl4 administration.The 8to 10-week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl4 in corn oil and sacrificed at the indicated time points.As a control, mice were left untreated.(A) At the indicated times, liver samples were taken for quantitative real-time PCR for cell-cycle genes (Ccnd1, Cdk4, Cdc25a, Cdk2, Ccnb1, Cdk1 and Cdk1n1a).The dotted line indicates expression levels in non-treated control animals.(B) Histological staining of the liver section for Ki67.Representative pictures are shown at 200x magnification.(C) The percentage of Ki67-positive hepatocytes are shown as % Ki67positive hepatocyte stained cells per field of view.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate mean ± SEM.

Figure 4 .
Figure 4. Cell-cycle gene expression in LysM-Cre Smad7 fl/fl animals after CCl 4 administration.The 8to 10-week-old Smad7 fl/fl and LysM-Cre Smad7 fl/fl littermates were injected with 30% CCl 4 in corn oil and sacrificed at the indicated time points.As a control, mice were left untreated.(A) At the indicated times, liver samples were taken for quantitative real-time PCR for cell-cycle genes (Ccnd1, Cdk4, Cdc25a, Cdk2, Ccnb1, Cdk1 and Cdk1n1a).The dotted line indicates expression levels in non-treated control animals.(B) Histological staining of the liver section for Ki67.Representative pictures are shown at 200x magnification.(C) The percentage of Ki67-positive hepatocytes are shown as % Ki67-positive hepatocyte stained cells per field of view.Data shown are representative of three independent experiments with three to five animals per group.Error bars indicate mean ± SEM.Statistical significance was calculated by ANOVA, but no significant p-values (≤0.05) between LysM-Cre pos and Cre neg Smad7 fl/fl were present in the data from panels A and C.