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. 2008 Oct;10(10):2067-77.
doi: 10.1111/j.1462-5822.2008.01189.x. Epub 2008 Jun 28.

The TLR2-MyD88-NOD2-RIPK2 signalling axis regulates a balanced pro-inflammatory and IL-10-mediated anti-inflammatory cytokine response to Gram-positive cell walls

Lilian O Moreira  1 , Karim C El Kasmi, Amber M Smith, David Finkelstein, Sophie Fillon, Yun-Gi Kim, Gabriel Núñez, Elaine Tuomanen, Peter J Murray
Affiliations

The TLR2-MyD88-NOD2-RIPK2 signalling axis regulates a balanced pro-inflammatory and IL-10-mediated anti-inflammatory cytokine response to Gram-positive cell walls

Lilian O Moreira et al. Cell Microbiol. 2008 Oct.

Abstract

Systemic infection with Streptococcus pneumoniae is associated with a vigorous pro-inflammatory response to structurally complex cell wall fragments (PnCW) that are shed during cell growth and antibiotic-induced autolysis. Consistent with previous studies, inflammatory cytokine production induced by PnCW was dependent on TLR2 but independent of NOD2, a cytoplasmic NLR protein. However, in parallel with the pro-inflammatory response, we found that PnCW also induced prodigious secretion of anti-inflammatory IL-10 from macrophages. This response was dependent on TLR2, but also involved NOD2 as absence of NOD2-reduced IL-10 secretion in response to cell wall and translated into diminished downstream effects on IL-10-regulated target gene expression. PnCW-mediated production of IL-10 via TLR2 required RIPK2 a kinase required for NOD2 function, and MyD88 but differed from that known for zymosan in that ERK pathway activation was not detected. As mutations in NOD2 are linked to aberrant immune responses, the temporal and quantitative effects of activation of the TLR2-NOD2-RIPK2 pathway on IL-10 secretion may affect the balance between pro- and anti-inflammatory responses to Gram-positive bacteria.

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Figures

Fig. 1
Fig. 1. Combined signals from TLR2 and NOD2 are required for PnCW-induced IL-10 production
A. Schematic representation of the composition of PnCW obtained by biochemical purification from whole lysed pneumococci. The upper part of the figure is cartoon of the bacteria cell membrane and wall indicating that the wall composed primarily of peptidoglycan, to which multiple proteins, lipopeptides and lipids are tethered, surrounds the cell membrane of pneumococci. Following purification, PnCW is predominantly peptidoglycan and teichoic acids. This fraction is particulate is nature, analogous to zymosan fractions from yeast cell walls. B. IL-10 production from BMDMs stimulated with PnCW measured over time by ELISA. Results are combined from independent experiments (n = 7). C. IL-10 production from LPS stimulation of BMDMs from the same genotypes used in (B). D. Northern blotting analysis of IL-10 mRNA over time (h) following PnCW stimulation of BMDMs from the indicated genotypes. Data from Nod2−/−; Tlr2−/− BMDMs were obtained from a different part of the gel as shown the separation from the other part of the gel. E. qRT-PCR analysis of IL-10 mRNA production following PnCW stimulation. Data points are averages of duplicate samples. Data are representative of three independent experiments. F. Confocal microscopy to follow the fate of PnCW after contact with BMDMs. FITC-labelled PnCW was added to C57BL/6 BMDMs and followed over time. Representative images at times 0, 5, 24 and 48 h are shown. At each time, cultures were fixed and labelled with phalloidin to visualize actin. Note that the FITC signal is not visible until sufficient PnCW particle have been phagocytosed and concentrated inside macrophages (24 h). All images were collected using a 10× objective, except the 48 h time point (20×). G. Higher power image of PnCW inside BMDMs at 48 h (40× objective).
Fig. 2
Fig. 2. Absence of NOD2 has a limited effect on PnCW-induced inflammatory transcription
A. Select inflammatory targets induced by PnCW in control or NOD2-deficient BMDMs. Data for each target mRNA is plotted as the average difference signal intensity. Data are averages from independent (n = 4) Affymetrix arrays performed per genotype and per time. No significant differences (paired t-test) were detected between genotypes. Complete data sets for this experiment have been deposited in the GEO database. B. Expression of IL-10 target genes is affected in the absence of NOD2 function. Signal intensity for IL-10 (left most in the panel) and selected genes whose expression is dependent, in part, on autocrine-paracrine IL-10 production. *P < 0.05, paired t-test comparing 4 h time points between control and Nod2−/− BMDMs (n = 4).
Fig. 3
Fig. 3
Pro-inflammatory cytokine transcription in response to PnCW is dependent on TLR2. BMDMs from the indicated genotypes were stimulated with PnCW for the times shown. Cytokine mRNAs were measured by qRT-PCR. Data are representative of three independent experiments.
Fig. 4
Fig. 4. IL-10 expression in response to PnCW is dependent on the RIPK2 pathway
A. BMDMs from Ripk2−/− or Nod1−/−; Nod2−/− mice were stimulated with PnCW and IL-10 measured over time by ELISA. B. As in (A), but following LPS stimulation. C and D. Analysis of BMDMs from an independent Ripk2 mutant strain showing IL-10 and IL-10 mRNA produced in response to PnCW requires RIPK2. E and F. Reconstitution of Nod2−/− hematopoietic stem cells with human or mouse NOD2 cDNAs rescues IL-10 production in response to PnCW. Stem cells from Nod2−/− mice were infected with retroviruses containing hNOD2 or mNOD2 cDNAs and selected by cell sorting for YFP and plated into media containing CSF-1 to drive differentiation into macrophages. After differentiation, macrophages were stimulated with LPS or LPS and MDP to measure the MDP synergy with TLR4 signalling (E) or IFN-γ signalling (F) or IL-10 production after PnCW treatment (G). Data are representation of two independent infection studies. In the case of mNOD2, sufficient cells were obtained to perform the MDP-IFN-γ synergy assay only.
Fig. 5
Fig. 5. PnCW activation of BMDMs does not activate the ERK or p38 pathways but requires MyD88 signalling
A. C57BL/6 BMDMs were stimulated with Pam3CSK4, LPS or PnCW over time and ERK phosphorylation measured by immunoblotting for phosphorylated forms of ERK or reprobed for total ERK amounts. B. A similar experiment to (A) was performed using BMDMs from the genotypes indicated on the right. C. The same lysates from (B) were probed for phospho-p38. Note that PnCW does not activate ERK or p38 in any of these experiments. D. BMDMs from control, Nod2−/−, Tlr2−/− or MyD88−/− mice were stimulated with PnCW for 8 h and IL-10 measured by ELISA. Data are average ± SD from independent samples (n = 3). E. BMDMs were stimulated with MDP or MDP + IFN-γ overnight and nitrites measured by the Griess assay. Stimulation with IFN-γ alone did not produce detectable nitrites (data not shown).
Fig. 6
Fig. 6
Model of IL-10 production myeloid lineage cells (macrophages and DCs) stimulated by agents that activate TLR2, alone or in combination with other cell surface molecules.
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