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Dietary modulation of the microbiome affects autoinflammatory disease

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Abstract

The incidences of chronic inflammatory disorders have increased considerably over the past three decades1. Recent shifts in dietary consumption may have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2cmo mice, which express a homozygous Leu98Pro missense mutation in the Pombe Cdc15 homology family protein PSTPIP2 (proline-serine-threonine phosphatase interacting protein 2), spontaneously develop osteomyelitis that resembles chronic recurrent multifocal osteomyelitis in humans2,3,4. Recent reports demonstrated a crucial role for interleukin-1β (IL-1β) in osteomyelitis, but deletion of the inflammasome components caspase-1 and NLRP3 failed to rescue Pstpip2cmo mice from inflammatory bone disease5,6. Thus, the upstream mechanisms controlling IL-1β production in Pstpip2cmo mice remain to be identified. In addition, the environmental factors driving IL-1β-dependent inflammatory bone erosion are unknown. Here we show that the intestinal microbiota of diseased Pstpip2cmo mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2cmo mice that were fed a diet rich in fat and cholesterol maintained a normal body weight, but were markedly protected against inflammatory bone disease and bone erosion. Diet-induced protection against osteomyelitis was accompanied by marked reductions in intestinal Prevotella levels and significantly reduced pro-IL-1β expression in distant neutrophils. Furthermore, pro-IL-1β expression was also decreased in Pstpip2cmo mice treated with antibiotics, and in wild-type mice that were kept under germ-free conditions. We further demonstrate that combined deletion of caspases 1 and 8 was required for protection against IL-1β-dependent inflammatory bone disease, whereas the deletion of either caspase alone or of elastase or neutrophil proteinase 3 failed to prevent inflammatory disease. Collectively, this work reveals diet-associated changes in the intestinal microbiome as a crucial factor regulating inflammasome- and caspase-8-mediated maturation of IL-1β and osteomyelitis in Pstpip2cmo mice.

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Figure 1: Changes in diet limit the development of inflammatory bone disease in Pstpip2cmo mutant mice.
Figure 2: Alterations in commensal microbiota landscape that are associated with Pstpip2cmo-mediated osteomyelitic disease can be modified by changes in diet.
Figure 3: Microbiota-mediated regulation of IL-1β expression shapes inflammatory bone disease.
Figure 4: Compensatory processing of IL-1β by caspase-1 and caspase-8 in neutrophils drives inflammatory bone disease.

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Acknowledgements

We thank D. Chaplin, V. Dixit, R. Flavell, D. Green and C. Pham for the supply of mutant mice. We thank J. Kim for helping to acquire and analyse the micro-computed tomography data. We thank S. Olsen, D. Roeber and the Genome Sequencing Facility in the Hartwell Center at St Jude Children’s Research Hospital for performing metagenomics sequencing of 16S rRNA. M.L. is supported by grants from Ghent University (BOF 01N02313 and 01J11113) and the European Research Council (Grant 281600). L.V.W. is a postdoctoral fellow of the Fund for Scientific Research-Flanders. This work was supported by: the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health, under Award Number AR056296 (T.-D.K.); the National Cancer Institute, part of the National Institutes of Health, under Award Number CA163507 (T.-D.K.); the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, under Award Number AI101935 (T.-D.K.); and ALSAC.

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Authors and Affiliations

Authors

Contributions

J.R.L., M.L. and T.-D.K. designed the study; J.R.L., P.G., L.V.W., C.R.C. and S.R.B. performed experiments, and G.R.J. provided technical assistance. D.J.M. and R.A.C. analysed the 16S rRNA metagenomics data, and P.V. performed and analysed the histopathology data. J.R.L., M.L. and T.-D.K. analysed data and wrote the manuscript; T.-D.K. oversaw the project.

Corresponding author

Correspondence to Thirumala-Devi Kanneganti.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Placing Pstpip2cmo mice on a high-fat and cholesterol diet limits the development of inflammatory bone disease.

ae, Wild-type and Pstpip2cmo mutant mice were fed a LFD or HFD. Representative hind paw images (a) and representative pictures of popliteal lymph nodes (b) from wild-type, LFD Pstpip2cmo and HFD Pstpip2cmo mice at 12–14 weeks of age. c, d, Haematoxylin and eosin staining (original magnification, ×20) (c) and pathology scores (d) of tail samples from 12–14-week-old wild-type, LFD Pstpip2cmo and HFD Pstpip2cmo mice. Pathology scores were assigned in a blinded fashion by a veterinary pathologist based on the extent and severity of inflammation, osteolysis and osteogenesis. e, Representative immunostaining of neutrophils and macrophages in hind paw sections from 14–18-week-old Pstpip2cmo mice that were fed either a LFD or a HFD (original magnification, ×60). ***P < 0.001; Student’s t-test.

Extended Data Figure 2 Consumption of a HFD limits hyperinflammatory cytokine production in Pstpip2cmo mutant mice.

a, Wild-type and Pstpip2cmo mutant mice were fed a LFD or HFD for 12 weeks. Relative expression of Cxcl1 (wild type n = 8; LFD Pstpip2cmo n =  4; HFD Pstpip2cmo n =  9) and Il6 (wild type n = 11; LFD Pstpip2cmo n =  10; HFD Pstpip2cmo n =  8) in the hind paws was determined by qRT–PCR. The bar graphs depict combined data from two independent experiments. Data are shown as mean ± s.e.m. b, Wild-type and Pstpip2cmo mutant mice were fed a LFD or a HFD for 12 weeks and cytokines levels in the hind paws were determined by ELISA. Combined data are from two independent experiments. Each point represents an individual mouse, and the line represents the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test.

Extended Data Figure 3 Placing Pstpip2cmo mice on a HFD does not cause abnormal weight gain, intestinal inflammation or extraintestinal translocation of commensal bacteria.

a, b, Wild-type BALB/cJ and Pstpip2cmo mice were fed ad libidum a LFD or a HFD. Body weight was measured in age-matched female (a) and male (b) mice at 12–16 weeks of age. Each point represents an individual mouse and the line represents the mean ± s.e.m. Data were combined from three independent experiments. ce, Colon length (c), colitis score based on rectal bleeding and stool consistency (d) and representative haematoxylin and eosin-stained sections (original magnification, ×20) (e) of the intestinal tract of LFD- and HFD-fed Pstpip2cmo mice aged 14–18 weeks. f, Presence of commensal bacteria in the spleen, liver, mesenteric lymph nodes and bone of wild-type and diseased LFD-fed Pstpip2cmo mice was evaluated by Gram staining and 16S rDNA qPCR analysis of eubacteria.

Extended Data Figure 4 Dietary- and microbiota-associated factors influence the production of pro-IL-1β.

a, Footpad homogenates of 12–16-week-old wild-type, LFD-fed Pstpip2cmo and HFD-fed Pstpip2cmo mice were immunoblotted for IL-1β. Data are representative of three independent experiments. b, Footpads samples were collected from 10–14-week-old specific pathogen-free wild-type, germ-free wild-type and Pstpip2cmo × Il1b−/− mice and pro-IL-1β protein levels were determined by western blotting. c, CD45+ cells were isolated from the colons of germ-free wild-type mice and cells were left untreated or stimulated with LPS for 1 h. Relative Il1b mRNA expression levels were determined by qRT–PCR. Two biological replicates, with two technical replicates each.

Extended Data Figure 5 Co-housing does not alter disease progression in LFD-fed Pstpip2cmo mice.

a, Pstpip2cmo mice were treated with a cocktail of broad-spectrum antibiotics in their drinking water. Faecal samples were collected from wild-type (n = 5) and Pstpip2cmo mice that received either regular drinking water (n = 5) or antibiotics water (n = 11) 5–7 weeks later. Prevotella and Flexispira 16S rDNA copy numbers were quantified and normalized to total bacteria. The bar graphs depict the mean ± s.e.m. b, Faecal microbiota from diseased Pstpip2cmo mice was orally transplanted into wild-type mice (Pstpip2cmo microbiota wild type) and the incidence of inflammatory bone disease in control Pstpip2cmo and faecal transplantation mice was evaluated. c, d, Pstpip2cmo mice were singly housed or co-housed with wild-type (c) or Il1b-deficient Pstpip2cmo (d) mice. Clinical development of bone deformity and arthritic inflammation in hind paws and tails was monitored over time. **P < 0.01, ***P < 0.001; Student’s t-test.

Extended Data Figure 6 The neutrophil associated proteases elastase and proteinase 3 are not required for Pstpip2cmo-mediated bone disease.

a, Incidence of inflammatory bone disease in Pstpip2cmo, Pstpip2cmo × Elane−/−, Pstpip2cmo × Elane−/− Prtn3−/−and Pstpip2cmo × Il1b/− mice. b, Representative footpad images from wild-type, Pstpip2cmo, Pstpip2cmo × Elane−/−, Pstpip2cmo × Elane−/− Prtn3−/−and Pstpip2cmo × Il1b−/− mice.

Extended Data Figure 7 Combined deletion of RIPK3 and caspase-8 does not provide protection against Pstpip2cmo-mediated osteomyelitis.

a, Incidence of osteomyelitic bone disease in wild-type, Pstpip2cmo, Pstpip2cmo × Il1b−/− and Pstpip2cmo × Ripk3−/− mice. b, Representative isosurface micro-computed tomography images of hind paw samples from 12–18-week-old Pstpip2cmo, Pstpip2cmo × Ripk3−/− and Pstpip2cmo × Ripk3−/− × Casp8−/− mice. c, Representative haematoxylin and eosin-stained sections of inflammatory caudal vertebrae bone lesions in diseased Pstpip2cmo, Pstpip2cmo × Ripk3−/− and Pstpip2cmo × Ripk3−/− × Casp8−/− mice (original magnification, ×4 (top) and ×10 (bottom)). d, qRT–PCR analysis of Il1b expression in footpads of wild-type (n = 7), Pstpip2cmo (n = 7) and Pstpip2cmo × Ripk3−/− × Casp8−/− × Casp1−/− (n = 7) mice aged 12–16 weeks. Data are expressed as mean ± s.e.m. of combined data from two independent experiments. **P < 0.01, ***P < 0.001; Student’s t-test.

Extended Data Figure 8 Reduced pro-IL-1β expression and IL-1β maturation in neutrophils isolated from HFD-fed Pstpip2cmo mice.

a, Wild-type, Pstpip2cmo and Pstpip2cmo × Il1b−/− bone-marrow-derived macrophages were left untreated or were primed with LPS for 3 h followed by stimulation with ATP (30 min) or silica (12 h), and IL-1β processing was evaluated by western blot. Data are representative of three independent experiments. b, Western blotting for pro-IL-1β in untreated neutrophils that were purified from wild-type, LFD-fed Pstpip2cmo and HFD-fed Pstpip2cmo mice. Data are representative of two independent experiments. c, d, Neutrophils (c) or macrophages (d) from wild-type, LFD-fed Pstpip2cmo and HFD-fed Pstpip2cmo mice were left untreated, or primed with LPS for 3 h and then stimulated with ATP (30 min) or silica (12 h), and IL-1β processing was evaluated by western blotting. Data are representative of two independent experiments.

Extended Data Figure 9 Depletion of neutrophils in anti-Ly6G treated Pstpip2cmo mutant mice.

Wild-type and Pstpip2cmo mice received either PBS or 500 μg per mouse anti-Ly6G antibody (clone IA8) by intraperitoneal injection every 4–5 days starting at 6 weeks of age. ac, Two weeks after the first anti-Ly6G treatment, FACS analysis was performed on peripheral blood leukocytes (PBLs). a, Representative FACS plots of Gr-1 and CD11b expression on CD45.2+ gated cells. b, Enumeration of CD45.2+Gr-1hiCD11b+ neutrophils in equal volumes of peripheral blood. c, Numbers of T cells (CD45.2+TCRβ+), CD45.2+Gr-1CD11b+ monocytes/macrophages and CD45.2+Gr-1intCD11b+ cells in equal volumes of peripheral blood. Each point represents an individual mouse and the line represents the mean ± s.e.m. ***P < 0.001; Student’s t-test.

Extended Data Figure 10 Dietary modulation of the intestinal microbiota composition drives autoinflammatory osteomyelitis by setting pro-IL-1β levels available for maturation by caspases 1 and 8.

Proposed model highlighting how dysbiosis and processing of IL-1β by caspases 1 and 8 contribute to the development of inflammatory bone disease.

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Lukens, J., Gurung, P., Vogel, P. et al. Dietary modulation of the microbiome affects autoinflammatory disease. Nature 516, 246–249 (2014). https://doi.org/10.1038/nature13788

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