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.
Figure 1.UPR pathways in mammalian cells. From Ribeiro and O'Neal (2012): ER Stress in Chronic Obstructive Lung Diseases. Curr Mol Med. 12:872-82. Click here to view full-size image...
Our studies have revealed that Ca2+i responses to infectious/inflammatory stimuli are increased in CF epithelia due to an expansion of the ER Ca2+ stores. We have subsequently shown that the ER/Ca2+ store expansion contributes to airway hyperinflammation by amplifying Ca2+-dependent inflammatory responses and increasing the ER capacity for the epithelial synthesis of inflammatory mediators. The initial findings in CF epithelia have been extended to other diseased epithelia, since we have found that ER/Ca2+ stores are also expanded in inflamed, native primary ciliary diskynesia and COPD human airway epithelia (see below).
Figure 2.Native human CF and COPD bronchial airway epithelia exhibit up-regulation of ER Ca2+ stores. A: XBP-1 mRNA splicing in freshly isolated CF vs. normal bronchial epithelia. B: Expression of the ER Ca2+ store markers calreticulin and IP3 receptors in normal and CF native epithelia. C: Calreticulin expression in normal and COPD epithelia. Modified from Ribeiro and O’Neal (2012): ER Stress in Chronic Obstructive Lung Diseases. Curr Mol Med. 12:872-82. Click here to view full-size image...
Because the ER/Ca2+ 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+ 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 (α and β) 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+ store expansion is mediated by IRE1α-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 IRE1α-dependent XBP-1 mRNA splicing coupled with up-regulation of ER Ca2+ stores (Fig. 2). Up-regulation of ER Ca2+ stores is also a feature of native COPD human airway epithelia (Fig. 2). Further studies in our lab have also implicated airway epithelial inflammation with the activation of an additional UPR pathway mediated by activating transcription factor 4 (ATF4; Fig. 1). 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 is shown in Fig. 3.
Figure 3.Model linking airway epithelial infection/inflammation (step 1)-induced UPR activation and the resulting adaptive responses (step 2) mediated by XBP-1 and ATF4 that are relevant for airway epithelial inflammatory responses. From Ribeiro and Boucher (2010): Role of endoplasmic reticulum stress in cystic fibrosis-related airway inflammatory responses. Proc. Am. Thorac. Soc. 7:387-94. Click here to view full-size image...
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+ stores and buffer 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 Ca2+ uptake stimulates mitochondrial respiration-dependent ROS production, we have addressed whether a direct correlation between the magnitude of Ca2+i signals and the mitochondrial generation of ROS exists in inflamed airway epithelia. Our current findings suggest that the increased Ca2+i signals resulting from ER/Ca2+ store expansion couple to a larger Ca2+i-mediated mitochondrial generation of ROS in inflamed airway epithelia. These alterations are relevant to oxidative stress responses of inflamed airways.
Figure 4. Native human COPD bronchial airway epithelia exhibiting mucous cell metaplasia. The immunofluorescent stains of MUC5AC and the ER marker calreticulin are depicted in green and red, respectively. Image from C. Ribeiro. Click here to view full-size image...
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.
The latest 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 recently published in Mucosal Immunology that the IRE1 isoform, IRE1ß, is specifically expressed in mucous cells (Fig. 5) and is required for airway mucin production.
These findings offer the proof of concept that IRE1β is a novel therapeutic target for the mucus overproduction characteristic of CF, COPD and asthmatic airways.
Figure 5.IRE1β expression is up-regulated in mucous cells from native inflamed CF human bronchial epithelia. IRE1β immunostain in normal and CF human bronchial epithelia. From Martino et al (2012): The ER Stress Transducer IRE1β is Required for Airway Epithelial Mucin Production. Mucosal Immunol. Nov 21. doi: 10.1038/mi.2012.105. [Epub ahead of print]. Click here to view full-size image...
Using in vitro approaches and performing initial studies in mouse models and native human airway epithelia, our laboratory has pioneered the concept that activation of the UPR is relevant to the pathophysiology of airway inflammation. 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 are well suited for the scientific mission of the newly formed Marsico Institute for Lung Health at UNC, an integrated translational/clinical research center. For example:
- Targeting IRE1/XBP-1 activation-dependent ER/Ca2+ store expansion may lead to therapies aimed at controlling the excessive inflammation of CF airways via reduction of Ca2+-dependent transcription of inflammatory mediators and production of ROS.
- Understanding the protective role of ATF4 against oxidative stress and amino acid loss resulting from protein secretion during inflammation may result in therapeutic strategies involving targeted activation of the ATF4 pathway aimed at protecting airway epithelia against the inflammatory burden of diseased airways.
- Generating small molecule inhibitors of IRE1β may lead to novel therapeutics for the overproduction of mucus characteristic of CF, COPD and asthmatic airways. Recent studies yielding mechanistic insights of IRE1 activation suggest that IRE1 inhibitors could prove to be powerful pharmaceuticals.
a) Role of IRE1β in airway inflammatory models associated with mucous cell functions
b) Role of XBP-1 and ATF4 in airway epithelial inflammatory responses
c) Functional relevance of UPR pathways in alveolar macrophage-mediated airway inflammation
Studies in collaboration with investigators at the UNC CF Center:
a) Role of UPR pathways in airway inflammatory responses. Collaboration with Dr. Wanda O'Neal.
g) Airway inflammatory responses to Pseudomonas aeruginosa mediated by ER stress. Collaboration with Dr. Scott Randell.
Studies in collaboration with investigators from the UNC School of Pharmacy:
Generation of small molecule inhibitors of IRE1β as therapeutics for CF, COPD and asthmatic airways disease. Collaboration with William Janzen, Emily Hull-Ryde and Jacqueline Norris-Drouin.
Studies in collaboration with investigators outside UNC Chapel Hill:
Studies with mice exhibiting airway epithelial specific deletion of XBP-1. Collaboration with Dr. Laurie Glimcher, Weill Cornell Medical College, Cornell University.
Studies in collaboration with international investigators:
a) Role of IRE1β in airway inflammatory responses. Collaboration with Dr. David Ron, University of Cambridge, and Drs. Bart Lambrecht and Sophie Janssens, University Hospital, VIB, Ghent, Belgium.
b) Role of IRE1β mutants in the regulation of airway mucin production. Collaboration with Dr. Kenji Kohno, Nara Institute of Science and Technology (NAIST).
c) Mechanisms of airway epithelial inflammation utilizing the SMM model. Collaboration with Dr. Giulio Cabrini, University of Verona, Italy.
d) Role of airway epithelial inflammation in the regulation of CFTR expression. Collaboration with Dr. Margarida Amaral, University of Lisbon, Portugal.
Carla Maria Pedrosa Ribeiro, Associate Professor of Medicine, Joint Associate Professor of Cell Biology and Physiology
1993-1998 Postdoctoral Calcium Signaling,
National Institute of Environmental Health Sciences, Research Triangle Park,NC
1987-1992 Ph.D. Cell Biology and Renal Physiology,
Duke University, Durham, NC
1978-1981 Med. Student Medicine,
Federal University of Pernambuco, Recife, Brazil
NIH/NHLBI, Cystic Fibrosis Foundation, American Asthma Foundation, Cempra Pharmaceuticals, UNC-Chapel Hill, NC TraCS Institute
Publications: Current publications can be found on by searching "ribeiro cm". Dr. Carla MP Ribeiro at the Department of Medicine, Division of Pulmonary and Critical Care Medicine
Published Journal Cover Figures
1. Paradiso, A. M., Ribeiro, C. M. P., and Boucher, R. C. (2001): Polarized Signaling via Purinoceptors in Normal and Cystic Fibrosis Airway Epithelia. J. Gen. Physiol. 117: 53-67.
2. Ribeiro, C. M. P., Paradiso, A. M., Livraghi, A., and Boucher, R. C. (2003): The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia. J. Gen. Physiol. 122: 377-387.
3. 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.
Mary Braun Martino, Research Specialist and Laboratory Manager
Mary Martino with Carla Ribeiro.
Dr. Bob Lubamba, Postdoctoral Research Associate.
Carla Ribeiro, Mary Martino, and Bob Lubamba
Carla Ribeiro with Dr. Alessandra Livraghi-Butrico, Research Associate and former Ribeiro lab member.
Jack Allen, Intern Student, North Carolina School of Science and Mathematics Durham, NC
Campus Box #7248
The University of North Carolina at Chapel Hill
Chapel Hill, NC 27599