Induction of Pulmonary Granuloma Formation by Propionibacterium acnes Is Regulated by MyD88 and Nox2

Abstract

Sarcoidosis is characterized by noncaseating granulomas with an unknown cause that present primarily in the lung. Propionibacterium acnes, an immunogenic commensal skin bacterium involved in acne vulgaris, has been implicated as a possible causative agent of sarcoidosis. Here, we demonstrate that a viable strain of P. acnes isolated from a patient with sarcoidosis and instilled intratracheally into wild-type mice can generate pulmonary granulomas similar to those observed in patients with sarcoidosis. The formation of these granulomas is dependent on the administration of viable P. acnes. We also found that mice deficient in the innate immunity adapter protein MyD88 had a greater number and a larger area of granuloma lesions compared with wild-type mice administered P. acnes. Early after P. acnes administration, wild-type mice produced proinflammatory mediators and recruited neutrophils into the lung, a response that is dependent on MyD88. In addition, there was an increase in granuloma number and size after instillation with P. acnes in mice deficient in CybB, a critical component of nicotinamide adenine dinucleotide phosphate oxidase required for the production of reactive oxygen species in the phagosome. Myd88^-/- or Cybb^-/- mice both had increased persistence of P. acnes in the lung, together with enhanced granuloma formation. In conclusion, we have generated a mouse model of early granuloma formation induced by a clinically relevant strain of P. acnes isolated from a patient with sarcoidosis, and, using this model, we have shown that a deficiency in MyD88 or CybB is associated with impaired bacterial clearance and increased granuloma formation in the lung.

Keywords: Propionibacterium acnes; granulomas; sarcoidosis.

Figure 1.

Generation of early pulmonary granuloma model. Wild-type (Wt) mice were administered 10⁹ viable Propionibacterium acnes intratracheally and were killed after 9 days to generate a mouse model of sarcoid-like granuloma formation. Naive Wt mice were used as control mice. (A) Naive (top left) and P. acnes–administered Wt mouse lungs (bottom left) were removed, fixed, embedded, and examined for granulomas (black arrowheads) via hematoxylin and eosin staining at low magnification. Scale bars: 600 μm. At higher magnification in P. acnes–administered mice (right), epithelioid macrophages (red arrowhead) and lymphoid cells (blue arrowhead) can be observed. Scale bar: 200 μm. Granuloma (B) numbers and (C) area were quantified using pattern recognition software Spatially Invariant Vector Quantization (SIVQ) from three tissue sections 200 μm apart; each individual data point represents the average of those measurements per mouse. (D and E) Naive and P. acnes–administered lungs were also perfused with phosphate-buffered saline, digested into single-cell suspensions, analyzed for cell type composition via flow cytometry, and gated as described in Materials and Methods. The number of total viable (D) lymphoid cells and (E) myeloid cells is shown. Lymphoid cells are CD4⁺, CD8⁺ T cells, and B cells; myeloid cells are AM, TM/MO, DC, and PMN. Statistical significance was determined using unpaired t tests (B and C) and multiple t tests (D and E). *P < 0.05, ***P < 0.001, and ****P < 0.0001. AM, alveolar macrophages; DC, dendritic cells; PMN, neutrophils; TM/MO, tissue macrophages/monocytes.

Figure 2.

Viable P. acnes drives granuloma formation in Wt mice. Wt mice were administered 10⁹ viable or heat-killed P. acnes intratracheally and were killed after 9 days. Granuloma (A) number and (B) area from Wt mice were quantified using pattern recognition software SIVQ from three tissue sections 200 μm apart; each individual data point represents the average of those measurements per mouse. Statistical significance was determined using unpaired t tests. **P < 0.01. HK, heat killed; Pa, P. acnes.

Figure 3.

Increased granulomas in Myd88^−/− mice. Myd88^−/− mice were administered 10⁹ viable P. acnes intratracheally and were killed after 9 days to test the role of MyD88 in granuloma formation. Naive Myd88^−/− mice were used as control mice. (A) Naive (top left) and P. acnes–administered (bottom left) Myd88^−/− mouse lungs were removed, fixed, embedded, and examined for granulomas (black arrowheads) via hematoxylin and eosin staining at low magnification (left). Scale bars: 600 μm. At higher magnification in P. acnes–administered mice (right), epithelioid macrophages (red arrowhead) and lymphoid cells (blue arrowhead) can be observed. Scale bar: 200 μm. Granuloma (B) numbers and (C) area were quantified using pattern recognition software SIVQ from three tissue sections 200 μm apart; each individual data point represents the average of those measurements per mouse. (D and E) Naive and P. acnes–administered lungs from Myd88^−/− mice were perfused with phosphate-buffered saline, digested into single-cell suspensions, analyzed for cell type composition via flow cytometry, and gated as described in Materials and Methods. The number of total viable (D) lymphoid and (E) myeloid cells is shown. Lymphoid cells are CD4⁺, CD8⁺ T cells, and B cells; myeloid cells are AM, TM/MO, DC, and PMN. Statistical significance was determined using unpaired t tests (B), Mann–Whitney tests (C), and multiple t tests (D and E). **P < 0.01, ***P < 0.005, and ****P < 0.0001.

Figure 4.

P. acnes drives MyD88-dependent proinflammatory mediator production. Wt and Myd88^−/− mice were administered 10⁹ viable P. acnes intratracheally and were killed after (A) 3 days or (B) 6 hours to look at early cellular recruitment and proinflammatory mediator production. Single-cell suspension of lung digest was stained for myeloid cell types: AM, TM/MO, DC, and PMN. Six hours after P. acnes administration, the lungs were homogenized and analyzed for the level of CXCL1, CXCL2, and IL-1α in lung homogenate. (B) Background levels of naive lung homogenate were subtracted from each mouse genotype. Statistical significance was determined using multiple t tests (A) and two-way analysis of variance with Sidak’s multiple comparisons (B). ***P < 0.0005, ****P < 0.0001. CXCL, CXC chemokine ligand.

Figure 5.

Increased granulomas in Cybb^−/− mice. Cybb^−/− mice were administered 10⁹ viable P. acnes intratracheally and were killed after 9 days to test the role of CybB/Nox2 in granuloma formation. Naive Cybb^−/− mice were used as control mice. (A) Naive (top left) and P. acnes–administered (bottom left) Cybb^−/− mouse lungs were removed, fixed, embedded, and examined for granulomas (black arrowheads) via hematoxylin and eosin staining at low magnification (left). Scale bars: 600 μm. At higher magnification in P. acnes–administered mice (right), epithelioid macrophages (red arrowhead) and lymphoid cells (blue arrowhead), together with giant cells (green arrowhead), can be observed. Scale bar: 100 μm. Granuloma (B) numbers and (C) area were quantified using pattern recognition software SIVQ from three tissue sections 200 μm apart; each individual data point represents the average of those measurements per mouse. (D and E) Naive and P. acnes–administered lungs from Cybb^−/− mice were perfused with phosphate-buffered saline, digested into single-cell suspensions, analyzed for cell type composition via flow cytometry, and gated as described in Materials and Methods. The number of total viable (D) lymphoid and (E) myeloid cells is shown. Lymphoid cells are CD4⁺, CD8⁺ T cells, and B cells; myeloid cells are AM, TM/MO, DC, and PMN. Statistical significance was determined using unpaired t tests (B), Mann–Whitney tests (C), and multiple t tests (D and E). ***P < 0.005, and ****P < 0.0001.

Figure 6.

Increased P. acnes burden in Myd88^−/− and Cybb^−/− mice. (A) Wt, (B) Myd88^−/−, and (C) Cybb^−/− mice were administered 10⁹ viable P. acnes intratracheally and were killed after 9 days. Serial sections of P. acnes–administered mouse lungs were removed, fixed, embedded, and examined for granuloma formation (black arrowheads) via hematoxylin and eosin staining at low magnification (left upper panels) and PAB (right upper panels). PAB staining (brown) can be observed (red arrowheads, bottom panels). Three days after P. acnes administration, Wt, Myd88^−/−, and Cybb^−/− mouse lungs were homogenized, plated, and grown on Schaedler agar. (D) CFU were enumerated. Statistical significance was determined using the Kruskal–Wallis test with multiple comparisons. **P < 0.01, ****P < 0.0001. H&E, hematoxylin and eosin; ns, not significant; PAB, P. acnes-specific stain.