Dietary modulation of the microbiome affects autoinflammatory disease

Abstract

The incidences of chronic inflammatory disorders have increased considerably over the past three decades. Recent shifts in dietary consumption may have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2(cmo) 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 humans. 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 Pstpip2(cmo) mice from inflammatory bone disease. Thus, the upstream mechanisms controlling IL-1β production in Pstpip2(cmo) 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 Pstpip2(cmo) mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2(cmo) 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 Pstpip2(cmo) 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 Pstpip2(cmo) mice.

Extended Data Figure 1. Placing Pstpip2^cmo mice on a high fat and cholesterol diet limits the development of inflammatory bone disease

a-e, Wild-type (WT) and Pstpip2^cmo mutant mice were fed a lean fat diet (LFD) or a high fat and cholesterol diet (HFD). Representative hind paw images (a) and representative pictures of popliteal lymph nodes (b) from WT, LFD Pstpip2^cmo and HFD Pstpip2^cmo mice at 12-14 weeks of age. c-d, Hematoxylin and eosin (H&E) staining (c) and pathology scores (d) of tail samples from 12-14 week old WT, LFD Pstpip2^cmo and HFD Pstpip2^cmo 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 weeks old Pstpip2^cmo mice that were fed either a LFD or a HFD. (***P < 0.001; Student’s t-test)

Extended Data Figure 2. Consumption of a HFD limits hyperinflammatory cytokine production in Pstpip2^cmo mutant mice

a, WT and Pstpip2^cmo mutant mice were fed a lean fat diet (LFD) or a high fat diet (HFD) for 12 weeks. Relative expression of KC (WT n=8; LFD Pstpip2^cmo n=4; HFD Pstpip2^cmo n=9) and Il6 (WT n=11; LFD Pstpip2^cmo n=10; HFD Pstpip2^cmo n=8) in the hind paws was determined by qRT-PCR. The bar graphs depict combined data from two independent experiments. Data is shown as mean ± s.e.m. b, WT and Pstpip2^cmo 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 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 Pstpip2^cmo mice on a HFD does not cause abnormal weight gain, intestinal inflammation or extraintestinal translocation of commensal bacteria

a-b, WT BALB/cJ and Pstpip2^cmo 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. c-e, Colon length (c), colitis score based on rectal bleeding and stool consistency (d) and representative haematoxylin & eosin (H&E)-stained sections (e) of the intestinal tract of LFD- and HFD-fed Pstpip2^cmo mice aged 14-18 weeks. f, Presence of commensal bacteria in the spleen, liver, mesenteric lymph nodes and bone of WT and diseased LFD-fed Pstpip2^cmo 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 proIL-1β

a, Footpad homogenates of 12-16 weeks old WT, LFD-fed Pstpip2^cmo and HFD-fed Pstpip2^cmo mice were immunoblotted for IL-1β. Data are representative of three independent experiments. b, Footpads samples were harvested from 10-14 week old specific pathogen free (SPF) WT, germ-free (GF) WT and Pstpip2^cmoxIl1β^−/− mice and proIL-1β protein levels were determined by Western blotting. c, CD45⁺ cells were isolated from the colons of GF WT mice and cells were left untreated (UT) or stimulated with LPS for 1 hr. Relative proIl1β 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 of LFD-fed Pstpip2^cmo mice

a, Pstpip2^cmo mice were treated with a cocktail of broad-spectrum antibiotics in their drinking water (Abx). Fecal samples were collected from WT (n=5) and Pstpip2^cmo mice that received either regular drinking water (n=5) or Abx 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, Fecal microbiota from diseased Pstpip2^cmo mice was orally transplanted into WT mice (Pstpip2^cmo microbiota » WT) and the incidence of inflammatory bone disease in control Pstpip2^cmo and fecal transplantation mice was evaluated. c-d, Pstpip2^cmo mice were singly housed or co-housed with WT (c) or Il1β-deficient Pstpip2^cmo (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 Pstpip2^cmo-mediated bone disease

a, Incidence of inflammatory bone disease in Pstpip2^cmo, Pstpip2^cmoxElastase^−/−, Pstpip2^cmoxNePr3^−/− and Pstpip2^cmoxIl1β^−/− mice. b, Representative footpad images from WT, Pstpip2^cmo, Pstpip2^cmoxElastase^−/−, Pstpip2^cmoxNePr3^−/− and Pstpip2^cmoxIl1β^−/− mice.

Extended Data Figure 7. Combined deletion of RIP3 and Caspase-8 does not provide protection against Pstpip2^cmo-mediated osteomyelitis

a, Incidence of osteomyelitic bone disease in WT, Pstpip2^cmo, Pstpip2^cmoxIl1β^−/− and Pstpip2^cmoxRip3^−/− mice. b, Representative isosurface micro-computed tomography (micro-CT) images of hind paw samples from 12-18 week old Pstpip2^cmo, Pstpip2^cmoxRip3^−/− and Pstpip2^cmoxRip3^−/−xCasp8^−/− mice. c, Representative 4x and 10x H&E sections of inflammatory caudal vertebrae bone lesions in diseased Pstpip2^cmo, Pstpip2^cmoxRip3^−/− and Pstpip2^cmoxRip3^−/−xCasp8^−/− mice. d, qRT-PCR analysis of proIl1β expression in footpads of WT (n=7), Pstpip2^cmo (n=7) and Pstpip2^cmoxRip3^−/−xCasp8^−/−xCasp1^−/− (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 proIL-1β expression and IL-1β maturation in neutrophils isolated from HFD-fed Pstpip2^cmo mice

a, WT, Pstpip2^cmo and Pstpip2^cmoxIl1β^−/− bone marrow-derived macrophages were left untreated (UT) or were first primed with LPS for 3 hrs followed by additional stimulation with ATP (30 mins) or silica (12 hours) and IL-1β processing was evaluated by Western blot. Data are representative of three independent experiments. b, Western blotting for proIL-1β in untreated neutrophils that were purified from WT, LFD-fed Pstpip2^cmo and HFD-fed Pstpip2^cmo mice. Data are representative of two independent experiments. b-c, Neutrophils (b) or macrophages (c) from WT, LFD-fed Pstpip2^cmo and HFD-fed Pstpip2^cmo mice were left untreated (UT), or primed with LPS for 3 hrs and then stimulated with ATP (30 min) or silica (12 hours) 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 Pstpip2^cmo mutant mice

WT and Pstpip2^cmo mice received either PBS or 500 μg/mouse anti-Ly6G antibody (clone IA8) by intraperitoneal injection every 4-5 days starting at 6 weeks of age. a-c, Two weeks following the first anti-Ly6G treatment, FACS analysis was performed on peripheral blood leukocytes. a, Representative FACs plots of Gr-1 and CD11b expression on CD45.2⁺ gated cells. b, Enumeration of CD45.2⁺Gr-1^hiCD11b⁺ neutrophils in equal volumes of peripheral blood. c, Numbers of T cells (CD45.2⁺TCRβ⁺), CD45.2⁺Gr-1^-CD11b⁺ monocytes/macrophages and CD45.2⁺Gr-1^loCD11b⁺ 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 proIL-1β levels available for maturation by caspases 1 and 8

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

Figure 1. Changes in diet limit the development of inflammatory bone disease in Pstpip2^cmo mutant mice

a-d, Wild-type (WT) and Pstpip2^cmo mutant mice were fed a lean fat diet (LFD) or a high fat and cholesterol diet (HFD). a, Incidence of inflammatory bone disease. b-d, Representative isosurface micro-CT paw scans (b), H&E sections (c) and pathology scores (d) for hind paw samples from 12-14 week old WT, LFD Pstpip2^cmo and HFD Pstpip2^cmo mice. Each point represents an individual mouse, and the line represents the mean ± s.e.m. (***P < 0.001; Student’s t-test)

Figure 2. Alterations in commensal microbiota landscape that are associated with Pstpip2^cmo-mediated osteomyelitic disease can be modified by changes in diet

a-c, Fecal samples were collected from WT, LFD Pstpip2^cmo and HFD Pstpip2^cmo mice at 10-12 weeks of age and 16S rRNA metagenomic sequencing was conducted. a, Heat map of fold differences in relative abundance of commensal bacteria. b, Principal coordinated analysis (PCoA) plot of fecal microbiota. c, Heat map of the top 20 commensal genera and species that differ between LFD Pstpip2^cmo and HFD Pstpip2^cmo mice are presented. d, Prevotella 16S rDNA copy numbers in WT and Pstpip2^cmo mice before (pre-disease: 3-6 weeks of age) and after (diseased: 10-16 weeks of age) the development of osteomyelitis. Each point represents an individual mouse, and the line represents the mean ± s.e.m. Data are representative of four independent experiments. e, 16S rDNA analysis of Prevotella abundance. Data are representative of four independent experiments. (**P < 0.01, ***P < 0.001; Student’s t-test)

Figure 3. Microbiota-mediated regulation of IL-1β expression shapes inflammatory bone disease

a, qRT-PCR analysis of relative proIl1β expression in the footpads of 12-16 week old WT, LFD Pstpip2^cmo and HFD Pstpip2^cmo mice. Each point represents an individual mouse, and the line represents the mean ± s.e.m. Combined data from three independent experiments. b, Protein levels of IL-1β in the hind paws. Combined data from two independent experiments. c, Relative proIl1β mRNA expression levels in CD45⁺ cells isolated from the colons of specific pathogen free (SPF) and germ-free (GF) WT mice. Two biological replicates, with two technical replicates each. d-e, Pstpip2^cmo mice were treated with a cocktail of broad-spectrum antibiotics in their drinking water (Abx). d, qRT-PCR analysis of colonic Il1β expression levels from 12-14 week old Pstpip2^cmo mice that received either regular drinking water (n=15) or Abx water (n=9). e, Incidence of inflammatory bone disease. f-i, Young Pstpip2^cmo mice (3 wks old) received PBS or fecal microbiota from diseased LFD Pstpip2^cmo or disease-free HFD Pstpip2^cmo mice by oral transplantation. f, 16S rDNA analysis of Prevotella copy numbers. g, Incidence of inflammatory bone disease. h-i, Representative footpad images (h) and H&E micrographs (i). (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test)

Figure 4. Compensatory processing of IL-1β by caspase-1 and caspase-8 in neutrophils drives inflammatory bone disease

a, Incidence of osteomyelitic disease in Pstpip2^cmo, Pstpip2^cmoxCasp1^−/−, Pstpip2^cmoxRip3^−/−xCasp8^−/−, Pstpip2^cmoxCasp1^−/−xRip3^−/−xCasp8^−/− (TKO) and Pstpip2^cmoxIl1β^−/− mice over time. b, Representative H&E staining of hind paw sections. c, Western blot analysis of IL-1β regulation in the footpads of 10-12 week old Pstpip2^cmo, Pstpip2^cmoxCasp1^−/−, Pstpip2^cmoxRip3^−/−xCasp8^−/−, Pstpip2^cmoxCasp1^−/−xRip3^−/−xCasp8^−/− (TKO) and Pstpip2^cmoxIl1β^−/− mice. d-f, WT and Pstpip2^cmo bone marrow-derived macrophages (d) or neutrophils (e-f) were left untreated (UT) or were first primed with LPS for 3 hrs followed by stimulation with ATP (30 mins) or silica (12 hrs). d-e, Secretion of IL-1β was measured by ELISA. Bar graphs depict mean ± s.e.m. One out of three biological replicates, with two-three technical replicates each. f, Western blot analysis of IL-1β in WT, Pstpip2^cmo, Pstpip2^cmoxCasp1^−/− and Pstpip2^cmoxCasp1^−/−xRip3^−/−xCasp8^−/− (TKO) neutrophils. g-i, WT and Pstpip2^cmo mice received either PBS or anti-Ly6G antibody every 4-5 days starting at 6 weeks of age. g, Incidence of inflammatory bone disease. h-i, Representative footpad images (h) and tail H&E micrographs (i).