Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 1;8(8):817-29.
doi: 10.1242/dmm.020362. Epub 2015 Jun 4.

Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis

Thean S Chew  1 , Nuala R O'Shea  1 , Gavin W Sewell  1 , Stefan H Oehlers  2 , Claire M Mulvey  3 , Philip S Crosier  2 , Jasminka Godovac-Zimmermann  1 , Stuart L Bloom  4 , Andrew M Smith  5 , Anthony W Segal  1
Affiliations

Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis

Thean S Chew et al. Dis Model Mech. .

Abstract

Crohn's disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion and confirm our previous findings, we performed transcriptomic analysis in macrophages and identified a subgroup of individuals with CD who had low expression of the autophagy receptor optineurin (OPTN). We then clarified the role of OPTN deficiency in: macrophage cytokine secretion; mouse models of bacteria-driven colitis and peritonitis; and zebrafish Salmonella infection. OPTN-deficient bone-marrow-derived macrophages (BMDMs) stimulated with heat-killed Escherichia coli secreted less proinflammatory TNFα and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors, suggesting that TNFα is mis-trafficked to lysosomes via bafilomycin-A-dependent pathways in the absence of OPTN. OPTN-deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis, and showed reduced levels of proinflammatory TNFα in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality, compared with wild-type mice. Optn-knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans might increase the risk of developing CD.

Keywords: Crohn's disease; Cytokines; Escherichia coli; Macrophages; TNFα.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
OPTN is upregulated and colocalises with TNF at the Golgi complex upon bacterial stimulation in macrophages. (A) OPTN expression in MDMs stimulated with Pam3 (TLR2), LPS (TLR4) and HkEc compared to unstimulated (US) MDMs (n=10-23/group). (B) Immunoblot for OPTN in MDMs after TLR2, TLR4 and HkEc stimulation (n=4). (C) Subcellular fractions of HkEc-stimulated THP-1 cells were immunoblotted for OPTN and markers for the Golgi complex (golgin-245 and GM130), lysosomes (LAMP1) and cytosol (GAPDH). (D) Immunoblot for OPTN after immunoprecipitation of OPTN from THP-1 cells. (E) Confocal microscopy in an MDM (white outline) stimulated with HkEc for 4 h were stained for GM130 (i) and OPTN (ii). Single-stained GM130 Golgi complex (white arrowheads) and double-stained OPTN and GM130 (pink arrowheads) are visible (iv). (F) Confocal microscopy in an MDM (white outline) stimulated with HkEc for 4 h were stained for TNF (i) and OPTN (ii), which colocalised within the Golgi complex (iii). Single-stained OPTN vesicles (pink arrowheads) and TNF vesicles (white arrowheads) are visible (iv). (G) Quantification of TNF vesicles that colocalised with OPTN and EEA1 (n=8-16 cells/person, 2 persons performed). (H) Confocal microscopy in an MDM (white outline) stimulated with HkEc for 4 h were stained for TNF (i) and early endosome antigen 1 (EEA1) (ii). Double-positive peripheral vesicles (pink arrowheads) and single-positive TNF vesicles (white arrowheads) are shown (iv). Results shown are mean±s.e.m., quantified immunoblots are normalised to actin (**P<0.01 and ***P<0.001; one-way ANOVA and Bonferroni's test for multiple comparisons). All images were taken with a 63× oil-immersion objective. Scale bars: 20 µm.
Fig. 2.
Fig. 2.
OPTN-deficient BMDMs secrete lower levels of proinflammatory cytokines on bacterial challenge. (A) Tnf, Il6, Il10 and Cxcl1 gene expression in Optn+/+ and Optn−/− BMDMs after stimulation with HkEc was measured with qRT-PCR (n=5 mice/group over 5 experiments). (B) TNF, IL6, IL10 and CXCL1 cytokine release in Optn+/+ and Optn−/− BMDMs after stimulation with HkEc was quantified using a multiplex cytokine plate (n=5 mice/group over 5 experiments). (C) Confocal microscopy in BMDMs stimulated with HkEc for 4 h and stained for intracellular TNF were quantified using ImageJ (n=177-178 cells/genotype over 2 experiments). (D) Confocal microscopy in BMDMs stimulated with HkEc for 4 h and stained for intracellular TNF and EEA1 were quantified using ImageJ (n=62-66 cells/genotype over 2 experiments). (E) Representative TNF immunoblot of whole cell lysates from BMDMs exposed to HkEc in the presence of lysosomal inhibitors. (F) Quantification of immunoblots for TNF showed significantly less intracellular 26-kDa precursor TNF in the BMDMs from Optn−/− mice, which is normalised to wild-type levels on addition of lysosomal inhibitors monensin, NH4Cl or chloroquine (n=3-8 mice/genotype over 3-5 experiments). (G) Representative TNF immunoblot of whole cell lysates from naïve and HkEc-stimulated BMDMs in the presence of brefeldin A or bafilomycin A. (H) Quantification of immunoblots for TNF in the presence of brefeldin A shows undetectable levels of the 17-kDa secreted form of TNF, whereas bafilomycin A results in significantly higher levels of the 17-kDa secreted TNF in Optn−/− BMDMs (n=3-8 mice/genotype over 3-5 experiments). Results shown are mean±s.e.m., all immunoblots are normalised to actin (*P<0.05, and **P<0.01; two-tailed, unpaired t-test).
Fig. 3.
Fig. 3.
OPTN is protective against in vivo bacterial infection. (A) Optn+/+ and Optn−/− mice were injected with different quantities of live E. coli into their peritoneum (n=4-12 mice/genotype over 3 experiments). (B) Mice were tail bled at day 2 to measure serum TNF levels. (C,D) Peritoneal washouts were performed on naïve Optn+/+ and Optn−/− mice and at day 1 after E. coli inoculation into the peritoneum for flow cytometry of (C) CD11b+ F4/80+ macrophages and (D) Gr1+ neutrophils (n=7-13 mice/genotype over 2 experiments). (E) Representative flow cytometry of Gr1+ neutrophils in Optn+/+ and Optn−/− mice at day 1. Results shown are mean±s.e.m. (*P<0.05 and **P<0.01; two-tailed, unpaired t-test).
Fig. 4.
Fig. 4.
Citrobacter colitis results in greater mortality and lower levels of serum pro-inflammatory cytokines in OPTN-deficient mice. (A,B) Optn+/+ (grey) and Optn−/− (black) mice were gavaged with live Citrobacter rodentium and followed for 9 days to assess (A) weight loss and (B) mortality (n=29-31 mice/genotype over 5 experiments). (C) Serum TNF, (D) IL6 and (E) CXCL1 were measured in naïve mice, day 2 and day 9 after Citrobacter inoculation, in tail bleed and cardiac puncture serum (n=5 mice/group). Results shown are mean±s.e.m. (*P<0.05, **P<0.01 and ***P<0.001; two-tailed, unpaired t-test and logrank test).
Fig. 5.
Fig. 5.
OPTN-deficient mice recruit fewer neutrophils during the early acute phase of Citrobacter infection, resulting in a more severe colitis. (A) Large bowel from naïve mice, day 3 and day 9 after Citrobacter inoculation, were digested and flow cytometry for CD11b+ Gr1+ neutrophils was performed (n=10-11 mice/genotype over 3 experiments). (B) Representative flow cytometry plots for CD11b+ Gr1+ neutrophils in naïve mice, day 3 and day 9 after Citrobacter inoculation, are shown with percentage of neutrophils. (C) Flow cytometry for CD11b+ F4/80+ macrophages, (D) CD3+ T cells and (E) CD19+ B cells in large bowels from naïve mice, day 3 and day 9. (F) Representative image of haematoxylin and eosin (H&E)-stained naïve and day 9 after Citrobacter inoculation large bowel tissue (20× magnification, scale bar: 200 µm). (G) Blinded colitis scoring of H&E-stained large bowel sections at day 9 in Optn+/+ and Optn−/− mice (n=17-20 mice/genotype, over 2 experiments). Results shown are mean±s.e.m. (*P<0.05; two-tailed, unpaired t-test).
Fig. 6.
Fig. 6.
Schematic diagram of TNF trafficking in murine macrophages. TNF is translated in the endoplasmic reticulum (ER), transferred to the trans-Golgi network (TGN) for post-translational modification before packaging into vesicles, and is trafficked via early endosome antigen 1 (EEA1)-positive vesicles to the plasma membrane for secretion (black arrows). In the absence of OPTN, a greater proportion of TNF is trafficked and degraded via bafilomycin-A-dependent pathways (red arrows). Monensin, chloroquine and NH4Cl inhibit lysosomal function, whereas brefeldin A blocks ER-to-Golgi transfer.
See this image and copyright information in PMC

References

    1. Albagha O. M. E., Visconti M. R., Alonso N., Langston A. L., Cundy T., Dargie R., Dunlop M. G., Fraser W. D., Hooper M. J., Isaia G. et al. (2010). Genome-wide association study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget's disease of bone. Nat. Genet. 42, 520-524. 10.1038/ng.562 - DOI - PMC - PubMed
    1. Baumgart D. C. and Sandborn W. J. (2012). Crohn's disease. Lancet 380, 1590-1605. 10.1016/S0140-6736(12)60026-9 - DOI - PubMed
    1. Bedell V. M., Westcot S. E. and Ekker S. C. (2011). Lessons from morpholino-based screening in zebrafish. Brief. Funct. Genomics 10, 181-188. 10.1093/bfgp/elr021 - DOI - PMC - PubMed
    1. Belzil V. V., Daoud H., Desjarlais A., Bouchard J.-P., Dupré N., Camu W., Dion P. A. and Rouleau G. A. (2011). Analysis of OPTN as a causative gene for amyotrophic lateral sclerosis. Neurobiol. Aging 32, 555.e13-555.e14. 10.1016/j.neurobiolaging.2010.10.001 - DOI - PubMed
    1. Bond L. M., Peden A. A., Kendrick-Jones J., Sellers J. R. and Buss F. (2011). Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane. Mol. Biol. Cell 22, 54-65. 10.1091/mbc.E10-06-0553 - DOI - PMC - PubMed

Publication types

MeSH terms