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Infectious diseases and fibrotic disorders: Potential novel targets
Duitman, JanWillem
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Duitman, J. (2014). Infectious diseases and fibrotic disorders: Potential novel targets
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Download date: 17 Jun 2017
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General discussion and summary
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SUMMARY
Chapter 1 gives an overview of the functions of C/EBPδ that are currently known, focusing
on the role of C/EBPδ in inflammation, cell proliferation and apoptosis, which are the most
relevant processes for the studies presented in this thesis. The knowledge on C/EBPδ in
infectious disease specifically is extended in the different chapters that follow in part 1 of
this thesis. In chapter 2 the role of C/EBPδ during experimental pneumococcal pneumonia
has been studied. We show that C/EBPδ aggravates the outcome of pulmonary S. pneumoniae infection in a Platelet-Activating Factor Receptor (PAFR)-dependent manner. C/
EBPδ-/- mice were protected during pneumococcal pulmonary infection which was evident
from lower bacterial dissemination and increased survival. Subsequently, in chapter 3 we
investigated the role of C/EBPδ during K. pneumoniae-induced pneumonia to elucidate
whether C/EBPδ is also detrimental during gram-negative pneumonia. We show however
that C/EBPδ is beneficial during pulmonary infection with K. pneumoniae. Indeed, Klebsiella-induced mortality was significantly increased among C/EBPδ-/- mice and we hypothesize
that the lack of macrophage mobilization in C/EBPδ-/- mice is a key mechanism causing the
higher mortality rates. Shortly after publication of chapter 3 another study about the role
of C/EBPδ in pulmonary inflammation was published with seemingly contradictive results.
Chapter 4 discusses the different results on C/EBPδ of chapter 2 and 3 in the perspective
of the publication in this recent paper and deliberates about the possible role of C/EBPδ
during lung infection and inflammation. We conclude that C/EBPδ is an important player
in inflammation but its role seems complex and is far from understood and therefore we
should be careful to extrapolate murine data to human disease and to the subsequent
clinical implementation.
Chapter 5 aims to elucidate whether C/EBPδ plays an important role during E. coliinduced urinary tract infection and describes the effect of C/EBPδ deficiency in a murine
model for acute urinary tract infection. We show that C/EBPδ does not play an important
role during this infection as evident from similar bacterial clearance rates in both wildtype
and C/EBPδ-/- animals. The last chapter of part I focus on the role of C/EBPδ in renal fibrosis using a murine model. In this chapter (Chapter 6) we show that C/EBPδ is protective
during renal fibrosis as is evident from enhanced tubular injury, collagen deposition in the
interstitial area, and higher expression of transforming growth factor-β in C/EBPδ-/- mice as
compared to wildtype mice. Several possible mechanisms (e.g.: macrophage influx and the
balance in proliferation and apoptosis) responsible for this protective effect are investigated with no positive result however.
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GENERAL DISCUSSION AND SUMMARY
The second part of this thesis extents our knowledge on the role of different PARs during
fibrotic disease. Both PAR-1 and PAR-2 have been described to play an important role in
cellular processes leading to excessive matrix accumulation in different organs like (among
others) lung and liver (described in Chapter 7). Subsequently, Chapter 8 shows the results of
an experiment in which PAR-1 signaling was blocked before and after the induction of lung
fibrosis. Inhibiting PAR-1 signaling was effective in limiting the development of pulmonary
fibrosis, even when administration of the antagonist was started 7 days after induction of
fibrosis. The results of this chapter are highly interesting especially as pharmacological treatment of lung fibrosis even after the onset of fibrosis is clinically very relevant. In Chapter 9
we aimed to elucidate whether PAR-4 also contributes to pulmonary fibrosis. We observed
that wildtype and PAR-4-/- mice developed fibrosis, but there were no differences in the
severity of fibrosis or in the expression of fibrotic markers like collagen and fibronectin in
PAR-4-/- mice compared with wildtype controls. Overall, these results demonstrate that PAR-4
does not play a significant role during pulmonary fibrosis. Chapter 10 aims to assess the contribution of PAR-1 to the development of (burn)wound-induced dermal fibrosis and attempts
to elucidate the underlying mechanism that leads to the thickening of the skin upon PAR-1
activation. We show that PAR-1 contributes to the development of skin fibrosis in a mouse
model of bleomycin-induced skin fibrosis as evident from reduced dermal thickening and
extracellular matrix (ECM) deposition in PAR-1-/- animals as compared to wildtype controls.
We suggest therefore that PAR-1 potentiates the fibrotic response by inducing fibroblast proliferation and ECM production. Next, in chapter 11, we aimed to elucidate the role of PAR-2
on the development of (burn)wound-induced skin fibrosis. Genetic ablation of PAR-2 exerted
potent antifibrotic effects and reduced dermal thickening, fibroblast proliferation and ECM
deposition during bleomycin-induced skin fibrosis. Using in vitro experiments we were able
to show that PAR-2 on HDF potentiates the fibrotic response by inducing proliferation and
ECM production and we hypothesize that these processes are the key mechanisms by which
PAR-2 contributes to skin fibrosis in vivo.
GENERAL DISCUSSION
In the first part of this thesis we intended to obtain further insight in the role of C/EBPδ in
the inflammatory response especially during infectious disease. C/EBPδ is generally accepted to act as a pro-inflammatory transcription factor and several studies show that C/EBPδ
may act as transcriptional activator of different pro-inflammatory cytokines. Indeed, in
response to LPS, administered either systemically or intranasally, C/EBPδ deficiency leads
to a dampened inflammatory response as evident from lower cytokine levels (e.g. IL-6 or
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TNF-α) as described, but not shown, in chapter 4 and reference 1 and 2.1;2 Although inflammation is a key process in the host response to bacterial infection, excessive and prolonged
exposure of different body tissues to inflammation can result in tissue damage, ultimately
leading to multiple organ failure and subsequent dead. Our finding that C/EBPδ is beneficial during Klebsiella pneumoniae-induced pulmonary infection suggests therefore that this
might be due to the regulation of the inflammatory response. Surprisingly, we did not find
a role for C/EBPδ in cytokine production that could be responsible for the observed effect,
but macrophage migration was however decreased in C/EBPδ-/- mice. On the other hand,
we show that the loss of C/EBPδ protects against infection by Streptococcus (S.) pneumoniae. While this result appears at odds with the aforementioned results, this particular
phenotype is explained by the role of C/EBPδ in lung epithelial PAFR, the receptor used
by S. pneumoniae to enter the organism, expression. Therefore, the relative resistance of
C/EBPδ deficiency to S. pneumoniae is unrelated to C/EBPδ’s role in the immune system
and no conclusions about the role of C/EBPδ in cytokine production can be drawn from
this study as the bacterial loads already skew the response to one side. Strikingly, also
during Escherichia (E.) coli-induced acute urinary tract infection no differences in cytokine
production were observed between C/EBPδ-/- mice as compared to wildtype mice. This is
particularly interesting as a previous study shows that IL-6 mRNA levels were reduced in C/
EBPδ-/- mice in response to E. coli-induced peritonitis.3 Lastly, during renal fibrosis, induced
by unilateral ureteral obstruction, we did not find a role for C/EBPδ in the inflammatory
response, as measured by macrophage infiltration or cytokine production.
Our results clearly show that the role of C/EBPδ in inflammation is complex and not as
straightforward as one might have thought. A possible explanation of the seeming contradictive results may reside in the fact that during bacterial infections different signalling
pathways are activated4-6, whereas previously reported results showing a role for C/EBPδ in
cytokine regulation are mainly obtained using single pathway activators (e.g.: LPS and IgG
Immune complexes).1;2;7 Knowledge about the role of C/EBPδ in the inflammatory response
derived from the studies described in this thesis have to be further extended, investigating
the seeming contradictive results on inflammation and to integrate results from different studies described elsewhere to get a better view on the precise role of C/EBPδ during
inflammation and infectious disease.
In the second part of this thesis we aimed to get more insight into the role of PARs in
fibrosis. Several studies already showed that PARs play a key role during fibroproliferative
disease of different organs. Indeed, genetic ablation of PAR-1 limits fibrosis in the lung8
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GENERAL DISCUSSION AND SUMMARY
and liver9, whereas PAR-2 deficiency protected against the development of lung10, liver11
and kidney12 fibrosis. The results described in this thesis show that PAR-1 and PAR-2 both
contribute to the fibrotic response in the skin and the role of these receptors in fibrosis
might well be part of a more general mechanism, rather than a tissue specific mechanism.
Interestingly, we also show that pharmacological inhibition of PAR-1 protected against
bleomycin-induced pulmonary fibrosis even when administered after the induction of the
fibrotic process. This is particularly of interest for the treatment of patients suffering from
pulmonary fibrosis as not many treatment options have proven to be highly effective.
Further studies are however needed before the inhibition of PAR-1 will be available as a
standard treatment option as all data described are obtained using murine models.
Although a potential role for PAR-4 in lung fibrosis has been suggested13;14, PAR-4 does not
seem to play an important role during the fibroproliferative response, at least for the lung.
Whether this holds also true for different organs remains elusive, but contribution of PAR4 in fibroproliferative disease is definitely not a general mechanism.
REFERENCES
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2. Yan, C., P. F. Johnson, H. Tang, Y. Ye, M. Wu, and H. Gao. 2012. CCAAT/Enhancer-Binding Protein delta Is a Critical Mediator of Lipopolysaccharide-Induced Acute Lung Injury. Am J Pathol.
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3. Litvak, V., S. A. Ramsey, A. G. Rust, D. E. Zak, K. A. Kennedy, A. E. Lampano, M. Nykter, I. Shmulevich, and A. Aderem. 2009. Function of C/EBPdelta in a regulatory circuit that discriminates
between transient and persistent TLR4-induced signals. Nat Immunol 10:437-443.
4. Balamayooran, T., G. Balamayooran, and S. Jeyaseelan. 2010. Toll-like receptors and NOD-like
receptors in pulmonary antibacterial immunity. Innate Immun 16:201-210.
5. Kovach, M. A. and T. J. Standiford. 2011. Toll like receptors in diseases of the lung. Int ImmunoPharmacol 11:1399-1406.
6. Opitz, B., V. van Laak, J. Eitel, and N. Suttorp. 2010. Innate Immune Recognition in Infectious and
Noninfectious Diseases of the Lung. Am J Respir Crit Care Med 181:1294-1309.
7. Yan, C., M. Zhu, J. Staiger, P. F. Johnson, and H. Gao. 2012. C5a-regulated CCAAT/Enhancer-binding Proteins beta and delta Are Essential in Fc gamma Receptor-mediated Inflammatory Cytokine
and Chemokine Production in Macrophages. J Biol Chem 287:3217-3230.
8. Howell, D. C. J., R. H. Johns, J. A. Lasky, B. Shan, C. J. Scotton, G. J. Laurent, and R. C. Chambers.
2005. Absence of proteinase-activated receptor-1 signaling affords protection from bleomycininduced lung inflammation and fibrosis. Am J Pathol 166:1353-1365.
9. Rullier, A., J. Gillibert-Duplantier, P. Costet, G. Cubel, V. Haurie, C. Petibois, D. Taras, N. DugotSenant, G. Deleris, P. Bioulac-Sage, et al. 2008. Protease-activated receptor 1 knockout reduces
experimentally induced liver fibrosis. Am J Physiol Gastrointest Liver Physiol 294:G226-G235.
10. Borensztajn, K., P. Bresser, C. van der Loos, I. Bot, B. van den Blink, M. A. den Bakker, J. Daal-
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huisen, A. P. Groot, M. P. Peppelenbosch, J. H. von der Thusen, et al. 2010. Protease-Activated
Receptor-2 Induces Myofibroblast Differentiation and Tissue Factor Up-Regulation during
Bleomycin-Induced Lung Injury Potential Role in Pulmonary Fibrosis. Am J Pathol 177:2753-2764.
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