PPARγ Deficiency Exacerbates Fibrotic Response to Mycobacteria Peptide in a Murine Model of Pulmonary Sarcoidosis

Malur, Anagha; Mohan, Anish; Barrington, Robert A.; Leffler, Nancy R.; Murray, Gina; Kew, Kimberly; Barna, Barbara P.; Thomassen, Mary Jane

doi:10.1164/ajrccm-conference.2019.199.1_meetingabstracts.a2429

Cited by 4 publications

(23 citation statements)

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“…Briefly, mice were sedated with isofluorane and by gently pulling forward the mouse tongue the epiglottis was exposed and a 50 μl volume was instilled using a pipette. MWCNTs (900–1201, lot-GS1802, SES Research, Houston, TX, United States) were freshly prepared and have been described previously ( 16 ). Sham controls received vehicle alone.…”

Section: Methodsmentioning

confidence: 99%

“…Paraffin embedded slides were sectioned at 7 μm, and stained with hematoxylin and eosin (H&E) or Gomori’s trichrome stain as previously described ( 4 , 16 ). A previously described semiquantitative scoring system ( 7 , 16 , 17 ) was used to calculate a relative comparison of the numbers and quality of granulomas formed in MWCNT-instilled mice. Trichrome stain was evaluated using modified Ashcroft method ( 16 , 18 ).…”

Section: Methodsmentioning

confidence: 99%

“…A previously described semiquantitative scoring system ( 7 , 16 , 17 ) was used to calculate a relative comparison of the numbers and quality of granulomas formed in MWCNT-instilled mice. Trichrome stain was evaluated using modified Ashcroft method ( 16 , 18 ).…”

Section: Methodsmentioning

confidence: 99%

“…Tracheobronchial lymph node volume, identified based on Van den Broeck et al, was determined using the formula (Length × Width 2 )π/6, as previously described ( 16 ). Lymph nodes were fixed overnight in PBS-buffered 10% formalin and paraffin embedded.…”

Section: Methodsmentioning

confidence: 99%

“…BAL cells were obtained as previously described ( 15 , 16 ). Total cell counts and differential counts were evaluated ( Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

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Matrix Metalloproteinase-12 Is Required for Granuloma Progression

et al. 2020

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Background Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by granuloma formation. Mechanisms for chronic persistence of granulomas are unknown. Matrix Metalloproteinase-12 (MMP12) degrades extracellular matrix elastin and enables infiltration of immune cells responsible for inflammation and granuloma formation. Previous studies report increased MMP12 in sarcoidosis patients and association between MMP12 expression and disease severity. We also observed elevated MMP12 in our multiwall carbon nanotube (MWCNT) murine model of granulomatous inflammation. Here we hypothesized that MMP12 is important to acute and late phases of granuloma pathogenesis. To test this hypothesis, we analyzed granulomatous and inflammatory responses of Mmp12 knock-out (KO) mice at 10 (acute) and 60 days (late) after MWCNT instillation. Methods C57BL/6 (wildtype) and Mmp12 KO mice underwent oropharyngeal instillation of MWCNT. Lungs were harvested at 3, 10, 20, and 60 days post instillation for evaluation of MMP12 expression and granulomatous changes. Bronchoalveolar lavage (BAL) cells were analyzed 60 days after MWCNT instillation for expression of mediators thought to play a role in sarcoid granulomatosis: peroxisome proliferator-activated receptor-gamma (PPARγ), interferon-gamma (IFN-γ), and CCL2 (MCP-1). Results Pulmonary granuloma appearance at 10 days after MWCNT instillation showed no differences between wildtype and Mmp12 KO mice. In contrast, by 60 days after MWCNT instillation, Mmp12 KO mice revealed markedly attenuated granuloma formation together with elevated PPARγ and reduced IFNγ expression in BAL cells compared to wildtype. Unexpectedly, Mmp12 KO mice further demonstrated increased alveolar macrophages with increased CCL2 at 60 days. Conclusions The striking reduction of granuloma formation at day 60 in Mmp12 KO mice suggests that MMP12 is required to maintain chronic granuloma pathophysiology. The increased PPARγ and decreased IFNγ findings suggest that these mediators also may be involved since previous studies have shown that PPARγ suppresses IFNγ and PPARγ deficiency amplifies granuloma formation. Interestingly, a role of MMP12 in granuloma resolution is also suggested by increases in both macrophage influx and CCL2. Overall, our results strongly implicate MMP12 as a key factor in granuloma persistence and as a possible therapeutic target in chronic pulmonary sarcoidosis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…BAL cells were obtained as previously described ( 15 , 16 ). Total cell counts and differential counts were evaluated ( Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

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Matrix Metalloproteinase-12 Is Required for Granuloma Progression

et al. 2020

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Studies in a Murine Granuloma Model of Instilled Carbon Nanotubes: Relevance to Sarcoidosis

Barna

Malur

Thomassen

2021

IJMS

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Poorly soluble environmental antigens, including carbon pollutants, are thought to play a role in the incidence of human sarcoidosis, a chronic inflammatory granulomatous disease of unknown causation. Currently, engineered carbon products such as multiwall carbon nanotubes (MWCNT) are manufactured commercially and have been shown to elicit acute and chronic inflammatory responses in experimental animals, including the production of granulomas or fibrosis. Several years ago, we hypothesized that constructing an experimental model of chronic granulomatosis resembling that associated with sarcoidosis might be achieved by oropharyngeal instillation of MWCNT into mice. This review summarizes the results of our efforts to define mechanisms of granuloma formation and identify potential therapeutic targets for sarcoidosis. Evidence is presented linking findings from the murine MWCNT granuloma model to sarcoidosis pathophysiology. As our goal was to determine what pulmonary inflammatory pathways might be involved, we utilized mice of knock-out (KO) backgrounds which corresponded to deficiencies noted in sarcoidosis patients. A primary example of this approach was to study mice with a myeloid-specific knock-out of the lipid-regulated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) which is strikingly depressed in sarcoidosis. Among the major findings associated with PPARγ KO mice compared to wild-type were: (1) exacerbation of granulomatous and fibrotic histopathology in response to MWCNT; (2) elevation of inflammatory mediators; and (3) pulmonary retention of a potentially antigenic ESAT-6 peptide co-instilled with MWCNT. In line with these data, we also observed that activation of PPARγ in wild-type mice by the PPARγ-specific ligand, rosiglitazone, significantly reduced both pulmonary granuloma and inflammatory mediator production. Similarly, recognition of a deficiency of ATP-binding cassette (ABC) lipid transporter ABCG1 in sarcoidosis led us to study MWCNT instillation in myeloid-specific ABCG1 KO mice. As anticipated, ABCG1 deficiency was associated with larger granulomas and increased levels of inflammatory mediators. Finally, a transcriptional survey of alveolar macrophages from MWCNT-instilled wild-type mice and human sarcoidosis patients revealed several common themes. One of the most prominent mediators identified in both human and mouse transcriptomic analyses was MMP12. Studies with MMP12 KO mice revealed similar acute reactions to those in wild-type but at chronic time points where wild-type maintained granulomatous disease, resolution occurred with MMP12 KO mice suggesting MMP12 is necessary for granuloma progression. In conclusion, these studies suggest that the MWCNT granuloma model has relevance to human sarcoidosis study, particularly with respect to immune-specific pathways.

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The M2a Macrophage Phenotype Accompanies Pulmonary Granuloma Resolution in Mmp12 Knock-Out Mice Instilled with Multiwall Carbon Nanotubes

Ogburn

Bhalla

Leffler

et al. 2021

IJMS

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Sarcoidosis is a chronic disease with unknown etiology and pathophysiology, characterized by granuloma formation. Matrix Metalloproteinase-12 (MMP12) is an elastase implicated in active granulomatous sarcoidosis. Previously, we reported that oropharyngeal instillation of multiwall carbon nanotubes (MWCNT) into C57Bl/6 mice induced sarcoid-like granulomas and upregulation of MMP12. When Mmp12 knock-out (KO) mice were instilled with MWCNT, granuloma formation occurred 10 days post-instillation but subsequently resolved at 60 days. Thus, we concluded that MMP12 was essential to granuloma persistence. The aim of the current study was to identify potential mechanisms of granuloma resolution in Mmp12KO mice. Strikingly, an M2 macrophage phenotype was present in Mmp12KO but not in C57Bl/6 mice. Between 10 and 60 days, macrophage populations in MWCNT-instilled Mmp12KO mice demonstrated an M2c to M2a phenotypic shift, with elevations in levels of IL-13, an M2 subtype-regulating factor. Furthermore, the M2 inducer, Apolipoprotein E (ApoE), and Matrix Metalloproteinase-14 (MMP14), a promoter of collagen degradation, were upregulated in 60-day MWCNT-instilled Mmp12KO mice. In conclusion, alveolar macrophages express two M2 phenotypes in Mmp12KO mice: M2c at 10 days when granulomas form, and M2a at 60 days when granulomas are resolving. Findings suggest that granuloma resolution in 60-day Mmp12KO mice requires an M2a macrophage phenotype.

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PPARγ Deficiency Exacerbates Fibrotic Response to Mycobacteria Peptide in a Murine Model of Pulmonary Sarcoidosis

Cited by 4 publications

References 0 publications

Matrix Metalloproteinase-12 Is Required for Granuloma Progression

Matrix Metalloproteinase-12 Is Required for Granuloma Progression

Studies in a Murine Granuloma Model of Instilled Carbon Nanotubes: Relevance to Sarcoidosis

The M2a Macrophage Phenotype Accompanies Pulmonary Granuloma Resolution in Mmp12 Knock-Out Mice Instilled with Multiwall Carbon Nanotubes

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