The MUC1 mucin specifically downregulates the LRP3 inflammasome
Garrett Z Ng 1,2 and Philip Sutton 1,2#
1Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia 2Centre for Animal Biotechnology, Faculty of Veterinary and Agricultural Science, University of Melbourne, Parkville, Victoria 3010, Australia
#Correspondence: Associate Professor Philip Sutton, Mucosal Immunology, Murdoch Childrens Research Institute, The Royal Children’s Hospital, Parkville, Victoria 3052, Australia. [email protected] ; Ph: +61 3 9936 6751; Fax: +61 3 9936 6528
KEYWORDS: Muc1; IL 1β; inflammasome.
Competing interests: The authors have no conflicts to declare.
Format = Genes and Immunity Page limit = 3 4 typeset pages incl 150 word abstract. Results and discussion together
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ABSTRACT
MUC1 is a cell membrane associated mucin expressed ubiquitously on mucosal epithelia and immune cells that limits the inflammatory response to multiple pathogens. We have recently shown that MUC1 controls inflammation resulting from
Helicobacter pylori infection by suppressing IL 1β produced via the NLRP3 inflammasome. Here we demonstrate that MUC1 also regulates IL 1β induced by the
NLRP3 activating bacteria Haemophilus influenzae but not bacteria that activate other inflammasomes. Using purified ligands we further demonstrate that MUC1 regulation of NLRP3 is specific, as it has no effect on the NLRP1b, NLRC4 and AIM2 inflammasomes. This indicates a unique role for MUC1 in the regulation of NLRP3 activating bacterial infections.
I TRODUCTIO
MUC1 is a cell surface associated mucin that is ubiquitously expressed on multiple cell types, including epithelial and immune cells, that limits the inflammatory immune response to infection by multiple mucosal bacterial pathogens including
Campylobacter jejuni , Helicobacter pylori , Pseudomonas aeruginosa and
Haemophilus influenzae.1 4 MUC1 is best known as a component of the physical barrier that prevents or limits contact or colonisation by pathogens such as C. jejuni and H. pylori.1; 2; 5 In concordance with this, MUC1 polymorphisms that result in shortened molecules are associated with H. pylori gastritis and disease.6; 7 However, we have recently demonstrated that, surprisingly, the inflammation resulting from H. pylori infection is primarily dependent on haematopoietic expression of MUC1 and thus not due to its barrier properties.8
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The initial events following infection include the activation of pattern recognition receptors including Toll like receptors (TLRs) that, via distinct signalling pathways, culminate in the production of cytokines. Certain cytosolic receptors activate a proteolytic complex called an inflammasome that in turn activates zymogenic caspase 1 that then converts inactive IL 1β and IL 18 to their mature active forms.
The NLRP3 inflammasome receptor is activated by a large number of stimuli including H. pylori.9; 10 We recently demonstrated that MUC1 regulates H. pylori induced gastritis by suppressing IL 1β production via NLRP3.8 However, the extent to which MUC1 regulates other inflammasomes is unknown. Here we demonstrate that MUC1 inflammasome regulation is specific to the NLRP3 inflammasome.
MATERIALS A D METHODS
Bacterial culture
H. pylori SS1 11 and Helicobacter felis CS1 12 were cultivated as described previously. 13 H. influenzae type B (ATCC10211) and non typeable (ATCC49247) were grown on chocolate agar at 37°C, 5% CO 2. Staphylococcus aureus
(ATCC29213), Salmonella Typhimurium (NCTC13347), Listeria monocytogenes
(EGD) and P. aeruginosa (NCTC10662) were all grown on horse blood agar at 37°C.
Stimulation of macrophages and IL 1β quantification
Peritoneal macrophages from specific pathogen free wildtype and Muc1 -/- 129/SvJ mice (kindly provided by Sandra Gendler; housed in the Parkville Veterinary Science animal house, University of Melbourne)14 were collected by peritoneal lavage with 10 mL cold 2% FCS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in PBS and cultured overnight in RPMI 1640 with 10% FCS, 1X penicillin and streptomycin
(Gibco). The next day, macrophages were stimulated with bacteria in antibiotic free
3 media for 2 hours prior to the addition of antibiotics (penicillin, streptomycin and 50
g/mL gentamicin). Alternatively, macrophages were stimulated using E. coli LPS
(100 ng/mL; Sigma Aldrich, St Louis, MO, USA), Anthrax protective antigen and
Anthrax lethal factor (List Biological Laboratories, Campbell, CA, USA), ultrapure
Salmonella Typhimurium flagellin, MDP, nigericin (Invivogen, San Diego, CA,
USA), alum (prepared as a 20 mg/mL stock, by mixing 10 mL of 10% aluminium potassium sulphate with 22.8 mL of 0.25 M sodium hydroxide), ATP (Sigma Aldrich) or gDNA either alone or sequentially. Lipofectamine 2000 (4 L/mL; Thermo Fisher
Scientific) was used to deliver ligands intracellularly. IL 1β was quantified by ELISA as previously described.15
Statistics
Data were analysed by two sided one way ANOVA (SPSS statistics version 21, IBM,
Armonk, NY, USA) and presented as median (horizontal bar), interquartile range
(box) and 10 th and 90 th percentiles (bars).
RESULTS A D DISCUSSIO
We initially tested whether MUC1 regulates IL 1β production in response to pathogens known to activate different inflammasome complexes (Fig 1A). While H. pylori, H. influenzae and S. aureus are all currently understood to only activate the
NLRP3 inflammasome,10; 16; 17 P. aeruginosa activates the NLRC4 inflammasome, S.
Typhimurium activates both the NLRP3 and NLRC4 inflammasomes and L. monocytogenes activates the AIM2, NLRC4 and NLRP3 inflammasomes.18 20 The inflammasome activating ability of H. felis has not yet been described. Increased levels of IL 1β were only secreted by Muc1 -/- macrophages in response to H. pylori and H. influenzae and S. aureus (Fig 1A). To ensure the IL 1β detected was a result of
4 inflammasome activation and not simply pro IL 1β non specifically released upon cell death, we analysed the supernatants by western blotting (Fig 1B). We found that the majority of IL 1β in the supernatant was cleaved, indicating activation of an inflammasome (Fig 1B). We hypothesized that, as these bacteria were those that only activate the NLRP3 inflammasome, this difference was because MUC1 has a specific role in regulation of the NLRP3 inflammasome.
Using purified ligands known to activate specific inflammasome complexes, we examined the ability of MUC1 to regulate individual inflammasome pathways. No difference was noted between wildtype and Muc1 -/- macrophages stimulated with anthrax lethal toxin (NLRP1b inflammasome), flagellin (NLRC4) or dsDNA (AIM2)
(Fig 2A). However, Muc1 -/- macrophages secreted increased levels of IL 1β compared to wildtype macrophages when stimulated with cytosolic MDP, which activates
NLRP3 in mice (Fig 2A). We then primed cells with LPS and stimulated with nigericin, alum, ATP, flagellin or dsDNA. Muc1 -/- macrophages secreted increased levels of IL 1β in response to the NLRP3 ligands nigericin, alum and ATP, but not flagellin or dsDNA (Fig 2B). We also assessed lactate dehydrogenase (LDH) release as a correlate for cell death, a common outcome of inflammasome activation. We found that there was increased death in Muc1 -/- cells after stimulation with nigericin but not flagellin or dsDNA (Fig 2C). These data show that MUC1 specifically regulates IL 1β production and cell death via the NLRP3, but not the other inflammasomes.
The downregulation of lung inflammation in response to P. aeruginosa and H. influenzae infection is currently attributed to the ability of MUC1 to suppress TLR signalling.3; 4 It is unlikely that suppression of the NLRP3 inflammasome is involved in MUC1 protection against P. aeruginosa pathogenesis, as P. aeruginosa does not
5 activate NLRP3 and did not induce increased IL 1β secretion from Muc1 -/- cells.
However, as H. influenzae is known to activate the NLRP3 inflammasome 16 and induced increased IL 1β secretion from Muc1 -/- cells, it is likely that the suppressive effect of MUC1 with this infection is in part due to NLRP3 inflammasome regulation.
We previously demonstrated that MUC1 plays no role in regulating the severity of gastritis resulting from H. felis infection.21 This was surprising given the important role of MUC1 in H. pylori pathogenesis.2; 8 At the time the reason for this was unclear, as we believed that the protective effects of MUC1 were due to its physical barrier properties. Now, we believe that the reason for this is probably that MUC1 does not regulate IL 1β production in response to H. felis , perhaps indicating these bacteria either do not activate the NLRP3 inflammasome or also activate another inflammasome pathway.
The NLRP3 inflammasome is unique in that it is necessary for expression of
NLRP3 to be induced by the TLR signalling cascade and NF κB activation prior to activation. 22 We have recently demonstrated that MUC1 suppresses activation of both p65 (NF κB) and IRAK4 in response to LPS or H. pylori to inhibit NLRP3 expression. 8 As IRAK4 is one of the first intermediaries in the TLR signalling cascade, MUC1 must interact with either TLRs, MyD88 or IRAK4 directly at the cell membrane. This mechanism also explains the specificity for the NLRP3 inflammasome, as other inflammasome receptors are expressed constitutively and thus would not be subject to this regulation.
In summary, we have determined that MUC1 inflammasome regulation is specific to the NLRP3 inflammasome. Extending our findings with H. pylori , this observation may be of importance for understanding the role of MUC1 in protecting against the
6 pathogenesis of other bacteria that uniquely activate the NLRP3 inflammasome, including H. influenzae and S. aureus .
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Figure 1 H. influenzae WT S. Typhimurium P. aeruginosa non-typeable A Muc1 -/- H. influenzae H. pylori H. felis S. aureus L. monocytogenes type B 1000 ** 800 *
600 * ** # (pg/mL) β β β β 400 IL-1 200
0 MOI - 1 10 1 10 1 10 1 10 1 10 1 10 1 10 1 10 B media Hp Hi LPS + Muc1 + - + - + - nigericin
pro-IL-1 β (35 kDa)
cleaved IL-1 β (17 kDa)
Figure 1: MUC1 differentially regulates the IL 1β response to specific bacterial pathogens. (A) Wildtype (n=11) and Muc1 -/- (n=4) macrophages from individual animals were stimulated overnight with MOI 1 or 10 of Helicobacter pylori , Helicobacter felis , Haemophilus influenzae , Staphylococcus aureus , Salmonella Typhimurium, Listeria monocytogenes or Pseudomonas aeruginosa . Antibiotics were added after 2 hours. Co culture with H. pylori or H. influenzae resulted in significantly more IL 1β secretion by Muc1 -/- cells (*, p<0.05; **, p<0.01; ANOVA), while co culture with S. aureus also increased IL 1β production by Muc1 -/- cells, although this did not reach significance (#, p=0.06; ANOVA). (B) IL 1β in pooled supernatants from (A) was examined by western blot. Supernatant from macrophages stimulated with 100 ng/mL LPS for 6 hours then 3 M nigericin for 2 hours was used as a positive control. The IL 1β secreted in response to H. pylori (Hp) and H. influenzae type B ( Hi) was largely cleaved indicating these bacteria activate an inflammasome.
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WT Figure 2 Muc1 -/- A 1.5 4 3 1.0 (ng/mL) (ng/mL) 2 β β β β β β β β 0.5 1 IL-1 IL-1 0.0 0 PA (1 g/mL) - + - + + + + lipofectamine - + - + + + LF ( g/mL) -- 1 0.5 0.25 0.12 0.06 flagellin (ng/mL) - - 250 250 125 62 2.0 3 ** * * 1.5 2 (ng/mL) 1.0 (ng/mL) β β β β β β β β 1 0.5 IL-1 IL-1 0.0 0 lipofectamine - + - + + + lipofectamine - + - + + + dsDNA ( g/mL) - - 1 1 0.5 0.25 MDP ( g/mL) -- 2.5 2.5 1.2 0.6
B *** C 4 *** 4 8 ** ** 3 ** 3 6
(ng/mL) 2 (ng/mL) 2 4 β β β β β β β β LDH LDH release
1 1 (fold change) 2 IL-1 IL-1 0 0 0 LPS - + + + + LPS - + + LPS - + + nigericin ( M) -- 6 3 1.5 ATP (5 mM) - - + nigericin -- + 2 3 10 *** ** 8 2 6
(ng/mL) 1 (ng/mL) β β β β β β β β 1 4 LDH release (fold change)
IL-1 IL-1 2 0 0 0 LPS - + + + + LPS - + + + + LPS - + + + + alum ( g/mL) - - 500 250 125 lipofectamine - - + + + lipofectamine - - + + + flagellin - - - + - flagellin - - - + - dsDNA - - - - + dsDNA - - - - +
Figure 2: MUC1 regulates IL 1β responses specifically via the LRP3 inflammasome. (A) Wildtype and Muc1 -/- macrophages (n=5 from individual animals) were stimulated with anthrax lethal toxin (protective antigen (PA) plus lethal factor (LF)), flagellin, gDNA or MDP overnight. Lipofectamine 2000, used to deliver MDP, flagellin and dsDNA into the cell cytosol, had no effect alone on IL 1β secretion. Muc1 -/- cells secreted significantly more IL 1β than wildtype cells when stimulated with cytosolic MDP, an NLRP3 ligand (**p<0.01, *p<0.05, ANOVA). We have observed differential IL 1β secretion in response to MDP but not DNA in three independent experiments. (B) Macrophages were pulsed with LPS for 6 hours then stimulated with ATP (n=4), nigericin, alum (n=6), flagellin (125 ng/mL) or dsDNA (1 g/mL, n=5) for two hours. Lipofectamine 2000 was used to deliver flagellin and dsDNA into the cytosol. Muc1 -/- cells secreted significantly more IL 1β than wildtype
9 cells only in response to the NLRP3 ligands nigericin, ATP and alum (***p<0.001, **p<0.01, ANOVA). (C) Cell death was assessed by lactate dehydrogenase (LDH) release after stimulation with nigericin (3 M), flagellin (125 ng/mL) or dsDNA (1 g/mL, n=5). There was significantly more LDH release from Muc1 -/- cells than wildtype cells in response to the NLRP3 ligand nigericin but not flagellin or dsDNA (**p<0.01, ANOVA).
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ACK OWLEDGEME TS
Contributors: PS and MAM obtained funding. GZN and PS designed and analysed experiments. GZN performed the experiments. GZN and PS prepared the manuscript.
All authors revised and approved the final manuscript.
Funding: This work was supported by the Victorian Government’s Operational
Infrastructure Support Program and by Project Grant #GNT1046254 from the
National Health and Medical Research Council of Australia. PS is supported by
Research Fellowship from the NHMRC and GZN was partly supported by a
Melbourne Research Scholarship from The University of Melbourne.
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Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Ng, GZ; Sutton, P
Title: The MUC1 mucin specifically inhibits activation of the NLRP3 inflammasome
Date: 2016-04-01
Citation: Ng, G. Z. & Sutton, P. (2016). The MUC1 mucin specifically inhibits activation of the NLRP3 inflammasome. GENES AND IMMUNITY, 17 (3), pp.203-206. https://doi.org/10.1038/gene.2016.10.
Persistent Link: http://hdl.handle.net/11343/124179
File Description: Accepted version