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3. Results
3.1 The in vitro monocyte-based model of inflammation
To build an in vitro model of inflammation, blood monocytes from 3 individual healthy donors were exposed in culture to a temporal sequence of different micro- environmental conditions that mimic the in vivo development of the inflammatory reaction to a viral challenge. Monocytes were isolated from buffy coats by density gradient isolation of peripheral blood mononuclear cells (PBMC) and magnetic selection of CD14
+cells. The recovered cells were viable and >98% were monocytes, as judged by morphological examination on cytosmears and by flow cytometrical analysis of scattering and CD14 positivity. It should be noted that the percentage of CD14
dimCD16
+monocytes after magnetic purification fully reflected the percentage of the same monocyte subpopulation in total blood and in PBMC (about 8-12% of total monocytes, data not shown). Therefore, the monocyte population we used in our experiments is representative of the monocyte heterogeneity as present in the circulation. The experimental procedures were carried out in the presence of 5% human pooled AB serum (as opposed to autologous serum or plasma), in order to avoid a putative source of variability.
As shown in Figure 1, freshly isolated monocytes were exposed to the chemokine CCL2 for 2 h at 37°C, to represent the CCL2 driven efflux of inflammatory monocytes from circulation to the site of inflammation. At 2 h, monocytes were exposed to a mixture of viral inflammatory stimuli, to mimic the encounter of inflammatory monocytes with infectious agents at the tissue site of reaction, and the temperature was raised to 39°C (as in an inflamed tissue). The challenge mixture included synthetic polyuridines (CL097) to stimulate TLR7/8, CpG motifs recognized by TLR9, poly(I:C) that is the ligand of the cytoplasmic RNA helicases RIG-I and MDA-5, and the human recombinant adenovirus serotype 5 (AdV5).
Coating of tissue culture plates with collagen and fibronectin, to reproduce the presence of extracellular matrix in the tissue microenvironment, was avoided after preliminary experiments, due to its potent direct macrophage activation (probably
“seen” as a foreign entity). The development of the inflammatory reaction was
reproduced by keeping the temperature at 39°C until 14 h and by adding in sequence
IFN-"/! (at 3 h, representing the tissue/resident cell reaction, like virus-activated
DCs, fibroblasts and epithelial cells) and IFN-# (at 7 h, representing the reaction of
the later influx of NK cells and Th1 cells). To reproduce the destruction of the
inflammation-inducing infectious agent and the resolution of the inflammatory
response, at 14 h all the inflammatory stimuli were washed off, the temperature was
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brought down to 37°C and fresh medium containing IL-10 was added (representing the activation of anti-inflammatory mechanisms). As conclusive phase of the resolution, at 24 h monocytes were exposed to TGF-! to reproduce macrophage deactivation towards reestablishment of tissue integrity and homeostasis.
The model for bacterial-induced inflammation was identical except for the use of LPS as bacterial stimulus, and TNF-" instead of IFN-"/!.
Figure 1. Temporal sequence of the in vivo development of the viral inflammatory reaction (in brackets the bacterial model).
3.2 Gene expression of cytokines and their receptors
It is known that IL-1! and IL-18 production can be regulated at transcriptional or post-transcriptional level. To evaluate if the different protein levels during the time course of the inflammatory reaction are due to the regulation of gene expression, the mRNA levels of the cytokines has been analysed. The copy number of the target gene was normalized by the reference gene hypoxanthine-guanine phosphoribosyltransferase, HPRT. In each donor, the expression level of IL1B (the gene coding for IL-1!) is up-regulated during the inflammatory phase in the viral model, however the interindividual variability is such that when averaging the data this increase is not statistically significant (Figure 2 left).
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Figure 2. IL1B gene expression levels (left: viral model; right: bacterial model)
In the bacterial model the level of IL1B is significantly up-regulated during the early inflammatory phase and decreases at 14 h and at 24 h (Figure 2 right).
However, it seems that the viral stimulation is stronger in inducing the expression of IL-1! as compared to LPS.
Figure 3. IL18 gene expression levels (left: viral model; right: bacterial model)
Expression of IL18 (the gene coding for IL-18) after the viral stimulation seems to be up-regulated early during the inflammatory reaction, but this increase again is not statistically significant when averaging the data of the three donors (Figure 3 left).
Conversely, LPS induce about a 4-fold increase of IL18 expression at 4 h, which then
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is down-regulated (Figure 3 right). The same down-regulation can be observed from 8 h also with viral stimulation, although not significant at all time points (Figure 3 left).
The expression of IL1RN (the gene coding for IL-1Ra) in the viral model has a peak at 4 h and it is significantly inhibited at 8 h, returning at baseline levels afterwards (Figure 4 left). In the bacterial model the IL1RN expression is down- regulated at 14 h (Figure 4 right).
Figure 4. IL1RN gene expression levels (left: viral model; right: bacterial model)
Expression of IL1R2 (the gene encoding IL-1RII) shows a bimodal profile in the
viral model, being up-regulated at 4 h and again at 24 h, i.e. during the crucial points
of the anti-inflammatory phase, although again this trend it is not statistically
significant (Figure 5 left). LPS stimulation does not affect the IL1R2 expression
(Figure 5 right).
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Figure 5. IL1R2 gene expression levels (left: viral model; right: bacterial model)
In the viral inflammatory reaction the expression levels of IL18BP (encoding IL- 18BPa) do not show significant changes over time (Figure 6 left), while on the contrary LPS induces strong expression of IL18BP at 14 and 24 h (Figure 6 right).
Figure 6. IL18BP gene expression levels (left: viral model; right: bacterial model)
3.3 Protein production and release after viral and bacterial stimulation
The IL-1! protein production by CD14
+monocytes has been evaluated in terms of
extracellular release of the mature protein at different time points during the
induction of viral and bacterial inflammation and the subsequent resolution of the
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reaction. The results show that IL-1! increase significantly at 14 h during the late inflammation, after treatment with viral stimuli and the IFNs (Figure 7 left). After washing of viral stimuli and the addition of IL-10 and TGF-!, the protein levels of IL-1! are significantly down-regulated. The IL-1! levels are expressed in terms of rate of release. In the graph it is possible to observe how the rate increases significantly until 14 h and then decreases during the resolution of inflammation. The rate of IL-1! protein release by monocytes stimulated with LPS has a similar trend, but with significant production already at 4 h and a lower peak production as compared to viral challenge (Figure 7 right).
Figure 7. Rate of release of the mature IL-1! protein (left: viral model; right: bacterial model)
In parallel to IL-1!, production of its inhibitors have been analysed. IL-1Ra is
released at high rate already at 4 h and this rate is sustained (and possibly increasing)
until 24 h, i.e. during the resolution phase (Figure 8 left). Then it significantly
decreases during the last part of the resolution of inflammation. Also in the bacterial
model IL-1Ra is produced/released at a very high rates already at 4 h and its
production remains sustained until resolution at 24 h, to decrease thereafter (Figure 8
right), in agreement with the behaviour in the viral model.
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Figure 8. Rate of release of the IL-1Ra protein (left: viral model; right: bacterial model)
The release of the soluble form of the IL-1RII (sIL-1RII) after viral stimulation has a peak during early inflammation (at 4 h), and it is significantly released until 24 h, to be abolished during the latest phase of resolution (Figure 9 left). The pattern of production of sIL-1RII by monocytes stimulated with LPS shows a highly significant release at 4 h, with no additional release during full inflammation (from 4 to 14 h) and a lower but measurable production during all the resolution phases (Figure 9 right).
Figure 9. Rate of release of sIL-1RII (left: viral model; right: bacterial model)
Another cytokine that has been analysed is IL-18. IL-18 activity does not depend
on the absolute levels of the cytokine, but rather on the relative amounts of IL-18 and
its inhibitor IL-18BP, reflecting the levels of free, and thereby active, IL-18. The
concentrations of free IL-18 have been therefore calculated, based on the measured
concentrations of both IL-18 and IL-18BP, and by applying the law of mass action
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knowing that the interaction is 1:1 and that the binding affinity between the two proteins is 400 pM [70]. Free IL-18 is released at significant levels during early inflammation (4 h), and is still substantial until 14 h in the virus-induced inflammation, while it is significantly down-regulated during the resolution phases (Figure 10 left). In monocytes stimulated with LPS, the level of free IL-18 has a peak at 4 h and is significantly produced, though at a much lower level, until 14 h, while practically absent in the resolution phases (Figure 10 right).
Figure 10. Rate of released free IL-18 (left: viral model; right: bacterial model)
Other than IL-1! and IL-18, the production of other inflammation-related
cytokines has been analysed. In the viral model, the rate of release of the
inflammatory cytokine IL-6 is maximal between 4 and 14 h (full inflammation) and
decreases during resolution (Figure 11 left). In the bacterial model, IL-6 production
follows a similar pattern but with higher levels (Figure 11 right).
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Figure 11. Rate of IL-6 production (left: viral model; right: bacterial model)
Another major inflammatory cytokine, TNF-", has been measured. Due to the design of the bacterial model, in which exogenous TNF-" was deliberately added to the cultures, the evaluation of the production of this cytokine by monocytes was possible only for the viral model. As shown in the Figure 12, TNF-" is strongly produced during all the inflammatory phases, and decreases during resolution down to undetectable.
Figure 12. Rate of TNF-" production in the viral model.
Two chemokines important in inflammatory reactions have been also analysed,
CXCL8 (IL-8) and CCL5 (RANTES). In the viral model, the rate of IL-8 production
is maximal during full inflammation, although already measurable in early
inflammation, decreases in the initial resolution phase (until 24 h), to disappear
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during late resolution (Figure 13 left). In the bacterial model, the rate of CXCL8 production is significant in all the reaction phases, being maximal during inflammation and decreasing during resolution (Figure 13 right).
Figure 13. Rate of CXCL8 production (left: viral model; right: bacterial model)
Viral stimulation induces CCL5 during early inflammation, with production decreasing but still high until 24 h, and going down significantly afterwards (Figure 14 left). In the bacterial model, CCL5 is strongly produced during the entire inflammatory phase (being maximal in the period between 4 and 14 h), but is still produced at very significant rates during the resolution phases (Figure 14 right).
Figure 14. Rate of CCL5 production (left: viral model; right: bacterial model)
