***Noteworthy: Dr. Ribeiro was one of three investigators honored by the UNC Eshelman School of Pharmacy as an innovator for work that helped lay the foundation to develop a new class of therapeutics for respiratory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Read the full story here.***
Specialty Areas: Inflammation Mechanisms in CF, COPD, and Asthma
Chronology: PhD: Duke University, 1992; Postdoctoral: NIH/NIEHS, 1993-1998; Research Associate, University of North Carolina, 1998-2001; Assistant Professor of Medicine, University of North Carolina, 2001-2010; Associate Professor of Medicine, University of North Carolina, 2010-present; Joint Associate Professor of Cell Biology and Physiology, University of North Carolina, July 2011-present.
Research Focus: Research in the Ribeiro laboratory focuses on studying mechanisms of airway inflammatory responses relevant to the pathogenesis of airway diseases characterized by mucus obstruction, inflammation, and oxidative stress, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. In particular, we study the functional roles of the endoplasmic reticulum (ER) and the mitochondria in the regulation of intracellular calcium (Ca2+i) signals and Ca2+i-mediated inflammation, and ER stress responses pertinent to the pathophysiology of these pulmonary diseases.
Techniques employed in the Ribeiro lab include:
- Primary culturing of human and murine airway epithelia; culturing of a variety of immortalized cell lines including airway epithelial cell lines.
- Bronchoalveolar lavages; isolation and culturing of murine and human alveolar macrophages.
- DNA, RNA and protein extraction from cells and tissues; DNA, RNA and protein quantitation; DNA cloning; RNA purification; RNase Protection Assays; PCR and RT-PCR; RNA microarrays.
- Northern and Southern blotting.
- Agarose and polyacrylamide gel electrophoresis; Western blotting.
- ELISA; immunocytochemistry; immunofluorescence; confocal microscopy.
- Measurements of intracellular calcium signals (including ER and mitochondrial calcium mobilization) by microfluorimetry and confocal microscopy.
- Assessment of intracellular reactive oxygen species.
- Assays to evaluate airway mucin production and secretion.
I joined the UNC Cystic Fibrosis Center in 1998 with 1) a strong background in calcium signaling, acquired during my postdoctoral training at the National Institute of Environmental and Health Sciences (NIEHS/NIH), and 2) a solid understanding of renal epithelial biology and membrane transport from my studies at Baylor College of Medicine and during graduate school at Duke University. Hence, the research in my lab has combined these areas of expertise to create a new field aimed at addressing fundamental questions in human airway epithelial biology involving Ca2+i-dependent responses and their role in airway inflammation.
Our studies have revealed that Ca2+i responses to infectious/inflammatory stimuli are increased in CF epithelia due to an expansion of the endoplasmic reticulum (ER) Ca2+i stores. We have subsequently shown that the ER/Ca2+i store expansion contributes to airway hyperinflammation by amplifying Ca2+i-dependent inflammatory responses and increasing the ER cpaacity for the epithelial synthesis of inflammatory mediators. The initial findings in CF epithelia have been extended to other diseased epithelia, since we have also found that ER/Ca2+i stores are also expanded in inflamed, native primary ciliary diskynesia and COPD human airway epithelia.
Because the ER/Ca2+i store expansion is a hallmark of several pulmonary diseases, we reasoned that it is an adaptive epithelial response that plays a key functional role in the pathophysiology of airway inflammation. We have addressed the mechanism responsible for the ER/Ca2+i store expansion during airway inflammation and found that it is mediated by activation of an ER stress response known as the unfolded protein response (UPR). In mammalian cells, activation of the UPR by increased levels of unfolded proteins in the lumen of the ER is sensed by 3 ER stress transducers, ATF6, IRE1 (a and b) and PERK. Activation of these UPR pathways results in downstream activation of signaling pathways relevant to the pathophysiology of airways diseases. (Fig. 1)
The UPR pathway responsible for the ER/Ca2+i stores is mediated by IRE1a-dependent mRNA splicing (activation) of the transcription factor X-box binding protein 1 (XBP-1). Indeed, native CF human airway epithelia exhibit up-regulation of IRE1a-dependent XBP-1 mRNA splicing coupled with up-regulation of ER/Ca2+i stores (Fig 2). Up-regulation of ER/Ca2+i stores is also a feature of native COPD human airway epithelia (Fig 2). Further studies in our lab have also implicated airway epithelia inflammation with the activation of an additional UPR pathway mediated by activating transcription factor 4 (ATF4). Activation of ATF4 is relevant to inflammatory responses, since ATF4 confers protection against oxidative stress, induces amino acid transport, and improves cellular metabolism and survival. Our current model for the roles of XBP-1 and ATF4 in airway epithelial inflammatory responses in shown in Figure 3.
The alterations in ER signaling resulting from activation of the UPR can have additional consequences for the cell biology of inflamed airway epithelia. For example, we have also reported that mitochondria are in close proximity to ER/Ca2+i stores and buffer ER/Ca2+i signals triggered by mucosal inflammatory mediators in human airway epithelia. Because mitochondrial respiration is a major source of intracellular reactive oxygen species (ROS), and mitochondrial ER/Ca2+i uptake stimulates mitochondrial respiration-dependent ROS production, we have addressed whether a direct correlation between the magnitude of ER/Ca2+i signals and the mitochondrial generation of ROS exists in inflamed airway epithelia. Our current findings suggest that the increased ER/Ca2+i signals resulting from ER/Ca2+i store expansion couple to a larger ER/Ca2+i mediated mitochondrial generation of ROS in inflamed airway epithelia. These alterations are relevant to oxidative stress responses of inflamed CF airways.
An important aspect of our research has been the development of a new model of CF airway epithelial inflammation, consisting of exposing normal airway epithelia to supernatant from mucopurulent material (SMM) from human CF airways. This model has been initially used to test the anti-inflammatory action of the macrolide antibiotic azithromycin, and has been subsequently used to test additional macrolides for anti-inflammatory effects. Please see below the additional studies we have initiated utilizing the SMM model.
A key feature of our research involving UPR activation and airway inflammation deals with a hallmark of chronic inflammation in CF, COPD and asthmatic airways, e.g., the overproduction of mucins (Fig 4). We have published in Mucosal Immunology that the IRE1 isoform, IRE1B, is specifically expressed in mucous cells (Fig. 5) and is required for airway mucin production. These findings offer the proof of concept that IRE1B is a novel therapeutic target for the mucus overproduction characteristic of CF, COPD and asthmatic airways.
Our latest published research has uncovered a key role for the IRE1a/XBP-1 pathway in mediating hyper-inflammatory responses of human CF alveolar macrophages.
We are currently investigating the functional role of additional branches of the UPR in other aspects of pulmonary inflammation, and in cigarette smoke-induced alterations in airway epithelial function. Our long-term goal is to establish the functional importance of UPR activation in airway inflammation by performing translational studies relevant to human airway diseases, including CF, COPD and asthma. These studies will likely lead to new therapies for these pulmonary diseases, based on targeting UPR pathways.
- O’Neal, W. K., Ribeiro, C. M. P. (2019): “Shocking the System” to Achieve Efficient Gene Targeting in Primary Human Airway Epithelia. Am J Respir Cell Mol Biol. [Epub ahead of print].
- Chen, G., Ribeiro, C. M. P., Sun, L., Okuda, K., Kato, T., Gilmore, R. C., Martino, M. B., Dang, H., Abzhanova, A., Lin, J. M., Hull-Ryde, E. A., Volmer, A. S., Randell, S. H., Livraghi-Butrico, A., Deng, Y., Scherer, P. E., Stripp, B. R., O’Neal, W. K., Boucher, R. C. (2019): XBP1S Regulates MUC5B in a Promoter Variant-Dependent Pathway in IPF Airway Epithelia. Am J Respir Crit Care Med. 15;200(2):220-234.
- Murphy, S. V., Ribeiro, C. M. P. (2019): Cystic Fibrosis Inflammation: Hyper-inflammatory, Hypo-inflammatory, or Both? Am J Respir Cell Mol Biol. 61(3):273-274.
- Gentzsch, M., Cholon, D. M., Quinney, N. L., Boyles, S. E., Martino, M. E. B., Ribeiro, C. M. P. (2018): The cystic fibrosis airway milieu enhances rescue of F508del in a pre-clinical model. Eur Respir J. 52(6).
- Webster, M. J., Reidel, B., Tan, C. D., Ghosh, A., Alexis, N. E., Donaldson, S. H., Kesimer, M., Ribeiro, C. M. P., Tarran, R. (2018): SPLUNC1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration. Eur Respir J. 52(4).
- Rimessi, A., Bezzerri, V., Salvatori, F., Tamanini, A., Nigro, F., Dechecchi, M. C., Santangelo, A., Prandini, P., Munari, S., Provezza, L., Garreau, de Loubresse N., Muller, J., Ribeiro, C. M. P., Lippi, G., Gambari, R., Pinton, P., Cabrini, G. (2018): PLCB3 Loss-of-function reduces P. aeruginosa-dependent IL-8 release in cystic fibrosis. Am J Respir Cell Mol Biol. 59(4):428-436.
- Abdullah, L. H., Coakley, R., Webster, M. J., Zhu, Y., Tarran, R., Radicioni, G., Kesimer, M., Boucher, R. C., Davis, C. W., and Ribeiro, C. M. P. (2018): Mucin production and hydration responses to mucopurulent materials in normal vs. CF airway epithelia. Am J Respir Crit Care Med. 197(4):481-491.
- Ribeiro, C. M. P., and Lubamba, B. A. (2017): Role of IRE1α/XBP-1 in Cystic Fibrosis Airway Inflammation. Int J Mol Sci. 18(1).
- Lubamba, B. A., Jones, L. C., O’Neal, W. K., Boucher, R. C. and Ribeiro, C. M. P. (2015): X-Box Binding Protein 1 and Innate Immune Responses of Human Cystic Fibrosis Alveolar Macrophages. Am J Respir Crit Care Med. 192(12):1449-61.
- Zhu, Y., Abdullah, L. H., Doyle, S. P., Nguyen, K., Ribeiro, C. M. P., Vasquez, P. A., et al. (2015) Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores. PLoS ONE. 10(5): e0127267.
- Okada, S F., Ribeiro, C. M. P., Sesma, J. I., Seminario-Vidal, L., Abdulah, L., van Heusden, C., Lazarowski, E. R., and Boucher, R. C. (2013): Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways. Am J Respir Cell Mol Biol. 49:814-20.
- Martino, M. E. B., Jones, L., Brighton, B., Ehre, C., Abdulah, L., Davis, C. W., Ron, D., O’Neal, W. K. and Ribeiro, C. M. P. (2013): The ER Stress Transducer IRE1β is Required for Airway Epithelial Mucin Production. Mucosal Immunol. 6: 639–654.
- Ribeiro, C. M. P. and O’Neal, W. K. (2012): ER Stress in Chronic Obstructive Lung Diseases. Curr Mol Med. 12:872-82.
- Ribeiro C. M. P. and Boucher RC. (2010): Role of endoplasmic reticulum stress in cystic fibrosis-related airway inflammatory responses. Proc. Am. Thorac. Soc. 7:387-94.
- Martino, M. E. B., Olsen, J. C., Fulcher, N. B., Wolfgang, M. C., O’Neal, W. K., and Ribeiro, C. M. P. (2009): Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1. J. Biol. Chem. 284:14904-13.
- Ribeiro, C. M. P., Hurd, H., Wu, Y., Martino, M. E. B., Jones, L., Brighton, B., Boucher, R. C., and O’Neal, W. K. (2009): Azithromycin Treatment Alters Gene Expression in Inflammatory, Lipid Metabolism, and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia. PLoS ONE Jun 5; 4(6):e5806.
- Livraghi A., Mall M., Paradiso A.M., Boucher R.C., and Ribeiro, C.M. P. (2008): Modeling dysregulated Na+ absorption in airway epithelial cells with mucosal nystatin treatment. Am. J. Respir. Cell Mol. Biol. 38:423-34.
- Ribeiro C. M. P., Paradiso, A. M., Schwab, U., Perez-Vilar, J., Jones, L., O’Neal, W., and Boucher, R. C. (2005): Chronic Airway Infection/Inflammation Induces a Ca2+i-dependent Hyperinflammatory Response in Human Cystic Fibrosis Airway Epithelia. J Biol Chem. 280:17798-17806.
- Ribeiro, C. M. P., Paradiso, A. M., Carew, M. A., Shears, S. B., and Boucher, R. C. (2005): Cystic fibrosis airway epithelial Ca2+i signaling. The mechanism for the larger agonist-mediated Ca2+i signals in human cystic fibrosis airway epithelia. J. Biol.Chem. 280:10202-10209.