Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (4), e18666

Inflammasome-independent Modulation of Cytokine Response by Autophagy in Human Cells

Affiliations

Inflammasome-independent Modulation of Cytokine Response by Autophagy in Human Cells

Tania O Crişan et al. PLoS One.

Abstract

Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient mice have shown that disruption of autophagy increases processing of IL-1β and IL-18 through an inflammasome-dependent manner. Using complementary approaches either inducing or inhibiting autophagy, we describe modulatory effects of autophagy on proinflammatory cytokine production in human cells. Inhibition of basal autophagy in human peripheral blood mononuclear cells (PBMCs) significantly enhances IL-1β after stimulation with TLR2 or TLR4 ligands, while at the same time reducing the production of TNFα. In line with this, induction of autophagy by starvation inhibited IL-1β production. These effects of autophagy were not exerted at the processing step, as inflammasome activation was not influenced. In contrast, the effect of autophagy on cytokine production was on transcription level, and possibly involving the inhibition of p38 mitogen activated protein kinase (MAPK) phosphorylation. In conclusion, autophagy modulates the secretion of proinflammatory cytokines in human cells through an inflammasome-independent pathway, and this is a novel mechanism that may be targeted in inflammatory diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Assessment of LC3 I and II levels in PBMCs under starving conditions or pharmacological treatment with 3MA.
Human PBMCs were pre-incubated for 1 hour at 37°C in either RPMI, starvation medium (EBSS) or EBSS with 3MA (10 mM) in which inhibitors of lysosomal fusion have been added: Ammonium chloride 20 mM and Leupeptine 100 µM. This was followed by 3 hours stimulation with culture medium, LPS (10 ng/ml) or Pam3Cys (10 µg/ml) prepared in the corresponding media (RPMI or EBSS). Cells were lysed and western blot of LC3 fractions I and II has been performed.
Figure 2
Figure 2. Modulation of inflammatory cytokine production by autophagy inhibition.
Freshly isolated human PBMCs were pre-incubated for 1 hour at 37°C in culture medium in the presence or absence of 3MA (10 mM) and afterwards stimulated with culture medium, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). After 24 hours incubation, IL-1β (A), TNFα (B) and IL-10 (C) were measured in the supernatant by specific ELISA. Data are presented as means ± SEM of cells harvested from 15 volunteers, **p<0.01, ***p<0.001.
Figure 3
Figure 3. The effects of 3MA on transcription and processing of inflammatory cytokines.
Cells pre-treated for 1 hour with culture medium with or without 3MA (10 mM) were stimulated for 4 hours with RPMI, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). RT-PCR was performed and relative levels of IL-1β and TNFα (B) mRNA were determined in 4 volunteers. (C) Western blot of p35 caspase-1 after 1 hour pre-incubation with or without 3MA (10 mM in RPMI), followed by 2 hours stimulation with LPS (10 ng/ml). The picture is representative for results obtained from 6 volunteers.
Figure 4
Figure 4. The influence of 3MA on the ATP-dependent release of IL-1β.
PBMCs were pre-incubated for 1 hour in the presence or absence of 3MA and were stimulated for 4 hours with LPS (10 ng/ml). After the stimulation, supernatants were discarded and refreshed with RPMI or with RPMI containing 1 mM ATP and cells were incubated for another 15 min. Data are shown as mean ± SEM of supernatant IL-1β levels obtained in 8 volunteers, **p<0.01.
Figure 5
Figure 5. The effects of starvation on mRNA levels of inflammatory cytokines.
Cells pre-incubated for 2 hours in either starvation medium (Earle's Balanced Salt Solution) or RPMI were stimulated for 4 hours with medium, LPS (10 ng/ml) or Pam3Cys (10 µg/ml) prepared respectively in starvation medium or RPMI. Subsequently, RT-PCR was performed and IL-1β mRNA levels are depicted as mean ± SEM of cells harvested from 6 volunteers, *p<0.05 (A). RT-PCR results of IL-1β (B) and TNFα (C) mRNA levels in cells pre-incubated for 2 hours with RPMI, starvation medium or starvation medium and 3MA (10 mM) followed by 4 hours stimulation with RPMI or LPS (10 ng/ml). Results are shown as mean ± SEM of data obtained in 4 volunteers.
Figure 6
Figure 6. MAPK influence on the 3MA-dependent modulation of IL-1β and TNFα production.
PBMC samples conditioned for 1 hour in either medium or in the presence of MAPK inhibitors: MEK inhibitor (5 µM), JNK inhibitor (20 µM) or p38 inhibitor (1 µM) were subjected to 1 hour treatment with RPMI or 3MA (10 mM), followed by stimulation with RPMI, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). After 24 hours incubation, specific ELISA was performed to determine the level of IL-1β in response to LPS (A) and Pam3Cys (B) stimulations, same as for TNFα (panels C and D, respectively). Data from 6 volunteers are shown as mean ± SEM. (E) Western blot of total and phosphorylated p38 in cells pre-treated for 1 hour with RPMI or 3MA (10 mM) and subjected to 1 hour stimulation with RPMI, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). (F) Quantification of the effect of 3MA on the phosphorylation of p38.

Similar articles

See all similar articles

Cited by 62 PubMed Central articles

See all "Cited by" articles

References

    1. Todde V, Veenhuis M, van der Klei IJ. Autophagy: Principles and significance in health and disease. Biochim Biophys Acta. 2009;1792:3–13. - PubMed
    1. Kundu M, Thompson CB. Autophagy: Basic Principles and Relevance to Disease. Annu Rev Pathol Mech Dis. 2008;3:427–55. - PubMed
    1. Hsieh YC, Athar M Chaudry IH. When apoptosis meets autophagy: deciding cell fate after trauma and sepsis. Trends Mol Med. 2009;15:129–138. - PMC - PubMed
    1. Alirezaei M, Kiosses WB, Flynn CT, Brady NR, Fox HS. PLoS ONE; 2008. Disruption of Neuronal Autophagy by Infected Microglia Results in Neurodegeneration. DOI: 10.1371/journal.pone.0002906. - DOI - PMC - PubMed
    1. Chen N, Karantza-Wadsworth V. Biochim Biophys Acta; 2009. Role and regulation of autophagy in cancer. DOI: 10.1016/j.bbamcr.2008.12.013. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback