Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. TFF-3 MODULATES RE- SPONSIVENESS TO BRON- CHODILATORS IN AIR- WAYS Mira Yin ✵ ABSTRACT Rhinovirus (RV) is the major cause of exacer- bations, or worsening of symptoms, in asthmatic chil- dren and adults. This often reduces the efficacy of therapeutic interventions such as bronchodilators — a type of medication used to promote airflow and alle- viate asthma symptoms. The exact mechanisms through which RV exposure decreases responsive- ness to bronchodilators remain unclear. Previous data demonstrates that airway cells release a specific sig- nature of inflammatory mediators following RV expo- sure. Other research has shown that Trefoil Factor 3 (TFF-3), one of the mediators identified by our screen, regulates cell motility in other cell types. We show that RV exposure attenuates relaxation in both the airway and human airway smooth muscle (HASM). Given our data, we aim to examine whether or not TFF-3 attenu- ates the relaxation of HASM and airways. Primary non-diseased human airway smooth muscle (HASM) was used to examine the conse- quences of TFF-3 in modulating HASM and airway re- laxation. Following RV-C15 exposure, it was found that the airway and HASM relaxation was attenuated. TFF-3 exposure also attenuated both airway and HASM relaxation. Additionally, TFF-3 exposure par- tially weakened iso-induced reversal of carbachol-in- duced phosphorylation of the myosin light chain. Within the cADDis Live Cell Assays, which provide real-time kinetic measurements of cyclic Adenosine Monophosphate (cAMP) production, TFF-3 attenu- ated formoterol-induced cAMP production. Research- ing how bronchodilation pathways change following RV infection can lead to the development of effective treatments and pharmaceutical solutions to alleviate worsening asthma symptoms during a viral exacerba- tion of the disease. 1 INTRODUCTION Rhinovirus, or RV, is the major cause of ex- acerbations in asthmatic children and adults and can lead to reduced efficacy of therapies for symptomatic relief. Previous research has found that RV produces unique effects on asthmatics in comparison to other viral infections, causing increased symptom severity and stronger effects on treatment response (Reddel et al. 2011). Another study revealed that bronchodilator treatments have reduced efficacy in both adult and pediatric patients once exposed to RV (James & Col- lins 2012). Although the mechanisms of RV induced asthmatic exacerbations are well-studied, research has yet to uncover exactly how RV may affect treat- ment efficacy. Figure 1: Proposed Trefoil Factor-3 Cell Signaling Pathway The proposed pathway for when TFF-3 is released into the body post-infection with rhinovirus, activat- ing cell receptors as a response. We identified one of the mediators released following RV infection of airway epithelial cells as Tre- foil Factor 3 (TFF-3). TFF-3 exhibits various properties, including forming dimers which interact with other pro- teins and aid in signaling within the body (Weste et al. 2022). Research has demonstrated that TFF-3 plays a role in the wound healing response and has estab- lished functions in mucosal cells in the gastrointestinal Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. tract, specifically aiding in their repair and regenera- tion post-damage (Braga et al. 2020) through en- hanced cell migration and anti-apoptotic effects (Fan et al. 2023). Given that TFF-3 engages in reparative re- sponses, it likely plays a similar role within the airways following RV exposure. Our findings suggest that TFF- 3 is present in a post-RV stimulation of primary human airway epithelial cells and intact in human lung slices. Figure 1 depicts a proposed signaling pathway by which TFF-3 can modulate airway function. As shown, the Human Airway Epithelial Cell (HAEC) releases lig- and TFF-3 post-exposure to RV, which activates Prote- ase-Activated Receptor-2 (PAR2) and/or C-X-C Motif Chemokine Receptor 4 (CXCR4) cell receptors on the human airway smooth muscle (HASM). Previous studies have established a connection between PAR2 and both inflammation and inflammatory diseases (Kennedy et al. 2020), while CXCR4 is related to tissue regeneration and cell proliferation. This in turn leads to the inhibition of alpha s, which is linked to the G protein and adenylyl cyclase (AC), decreasing cAMP production and airway relaxation. (Bianchi & Mezzapelle 2020). 2 MATERIALS AND METHODS Figure 2: Human Airway Smooth Muscle Analysis Methods (Left) Cells cultured on both sides of the transwell insert until confluency reached (Top Right) Western Blotting (Bottom Right) cAMP assay work- flow. This study utilized primary HASM cells to con- duct the various procedures seen in Figure 2. We ob- tained human lung tissue samples from the National Disease Research Interchange (NDRI) and from the In- ternational Institute for the Advancement of Medicine (IIAM). Primary HASM cell lines originated from the trachea of both healthy, non-asthmatic donors with no history of chronic diseases or smoking, and from dis- eased, aborted-transplant human lung donors. Cells were cultured in F-12 medium supplemented with 10% Fetal Bovine Serum (FBS) to encourage growth, along with 100 U/mL penicillin, 0.1 mg/ml streptomycin, and 0.1% Primocin from Invitrogen to prevent contamina- tion. To split the cell lines, we washed the cells with phosphate buffered saline (PBS) and aspirated the solu- tion. Then, we added the enzyme 0.05% 1X Trypsin- EDTA (Gibco) to detach and release the cells from the bottom of the plates or flasks. Later in the procedure, we added in feeding media to prevent the trypsin from lys- ing the cells and causing complete damage. The experiment utilized the HASM cells in subculture during passages 1–5, as these cells retain the expression of native contractile protein. HAEC was derived from the same tracheas as the HASM cells. Cells were on transwell inserts from Corning and were fed on both the upper and lower side of the insert with Pneumacult Ex Basal Media from StemCell Technolo- gies until they reached the appropriate level of con- fluency. The cells then underwent air-liquid interface (ALI) differentiation for 21 days prior to stimulation and were fed with Pneumacult ALI Media. We fed and maintained cell lines by routinely replacing media in order to prevent the cells from drying out. Figure 3: Western Blotting Media Treatments HASM underwent treatment to test the effects of TFF-3 on protein modifications that are surrogates for bronchodilation/relaxation. The cells grew to conflu- ence until the desired percentage of cells covered the surface of the plate, then were serum starved with stock media 24-74 hours before treatment. This Western blot Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. utilized four different cell donor lines, with cells receiv- ing a treatment of 0.15μM and 1.5μM of TFF-3 48 hours before short-time point treatments. After the 48-hour period, specific wells then received a treatment of either carbachol (carbamoyl choline - Cch) solution at 20μM for 10 minutes, a bronchodilator known as isoprenaline hydrochloride/isoproterenol (Iso) at 1μM for 5 minutes, or both (Figure 3). Sigma Aldrich (St. Louis, MO) sup- plied Carbachol (Cch), Iso, and perchloric acid. RNA transfection produced Rhinovirus C15, followed by a RNase A treatment of clarified lysates and ultracentrifu- gation through a 30% sucrose cushion to purify the vi- rus. Cell Signaling Technologies supplied Radio-Im- munoprecipitation Assay (RIPA) buffer, utilized to lyse the cells. A quick addition of stop buffer (perchloric acid) then halted intracellular signaling, followed by ly- sate collection for Western blotting. After collection, lysate either underwent soni- cation or freezing to break the cell membranes and release the contents, followed by a heating process at 70°C for 10 minutes to denature the samples. The samples then underwent centrifugation at 14000 rpm for 5 minutes. Using a micropipette, we added 3uL of the latter, 15uL of the samples, and 5uL of the sample buffer into 15 4-12% Bis-Tris polyacrylamide gel wells. After running the gels, iBlot facilitated the transfer of samples onto the membrane for staining with anti- bodies against phosphorylated myosin light chain (pMLC) and total myosin light chain (MLC) to compare the fold change of expression between the two. Anti- bodies for detection of pMLC came from Cell Signal- ing Technologies. EMD Millipore supplied the MLC antibodies. We then used ImageStudio to visualize membranes and analyze band intensities. Figure 4: Generation and Measurement of Human Precision Cut Lung Slices (Top) Method shown in images depicting how hPCLS are generated from human lung samples. (Bottom) hPCLS are then measured to measure con- traction (when Cch stimulated) and relaxation (when Iso stimulated). Human precision cut lung slices (hPCLS) shown in the top section of Figure 4 originated from the lungs of diseased or non-diseased patients and re- ceived an injection of 2% low melting agarose into the bronchi and branched airways to inflate them. After the agarose set in ice, we sectioned the lung lobes and isolated the airways into 8mm diameter cores us- ing the coring tool and sliced at a thickness of 350μm using the Precisionary Instruments VF300 Vibratome. The best intact airways provided samples for PCLS as- says and separate measurements for contraction and relaxation as shown in the bottom of Figure 4. Any other airway slices contributed to supernatant experi- ments. Muscle relaxation was tracked by measuring cAMP production. To measure the total cellular cAMP production in cell lysates, we plated sub-confluent HASM cells and allowed them to grow to ~80% conflu- ence using cAMP assays purchased from the Applied Bi- osystems cAMP-Screen Immunoassay System, adhering to the manufacturer provided instructions. After aspirat- ing the media, we replaced it with conditioned media from a control buffer or with RV-C15 stimulated Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. epithelial cells for 48 hours prior to stimulation with iso- proterenol. The cells underwent lysis and were incu- bated for 30 minutes at 37°C. We then added Conju- gate Dilution Buffer, cAMP-AP Conjugate, anti-cAMP antibody, and the samples to a pre-coated assay plate to incubate for 1 hour on a plate shaker. After a 30 mi- nute incubation period with CSPD®/Sapphire-II RTU Substrate, a luminometer measured light emission from the plates. We derived data from standard curves and reported cAMP levels following standard dilutions. When performed with the cell lines, the cAD- Dis Live Cell Assays detected Gs protein activation, which when stimulated produces cAMP. This allowed for the determination of TFF-3’s effect on this signal. This assay was able to measure cAMP production in real time, showcasing the response kinetics with greater dimming and indicating a higher cAMP con- centration in the cells. On Day 0, cells underwent preparation for transduction and seeding. We decanted T75 contain- ing confluent HASMCs by removing the media within the flask, and then we washed the cells with 1X PBS (Gibco). We then trypsinized the cells to release them from the bottom of the flask and isolated them for plating in F12 feeding medium supplemented with 10% FBS and Primocin (Gibco). We counted the cells in suspension using a hemocytometer and 0.4% Try- pan Blue (Gibco). Cell density varied per experimental target. Cells were plated in a 96 well black-walled tis- sue culture treated assay plate (Corning COSTAR). The cells grew and proliferated for two days until transduction. To perform the transduction, we supple- mented the media with a combination of the cADDis Green Down cAMP Sensor Assay (Montana Molecular) and Trichostatin A (Sigma). The cells incubated for an- other two days until imaging. We took images once per second using an inverted fluorescent microscope to compile a cohesive TIFF file that can later undergo analysis using Python for image analysis and Prism for generating time course curves. One minute into the sequence, we used formoterol fumarate dihydrate (Sigma) to invoke the cAMP response, leading to a downward shift in green fluorescence. 3 RESULTS Figure 5: TFF-3 Stimulation of HASM Partially Atten- uates Isoproterenol-Induced Reversal of Carbachol- Induced Phosphorylation of Myosin Light Chain (pMLC) HASM were stimulated with TFF-3, and then with Cch ± Iso, lysates collected for Western blot analysis. (Left) Immunoblot of pMLC and MLC from n=1 donor cell line (Right) Band density measurements of pMLC nor- malized to MLC, n=4 distinct donors Figure 5 depicts a representative immunoblot from one of the four cell lines utilized within the experi- ments. The red shows the total MLC and the green shows the phosphorylated MLC. The group mean data from Figure 5 shows the average fold change be- tween the four cell lines used in the experiments, indi- cating minimal change in the pMLC signal in the Cch + TFF-3 treatment. Compared to the Cch + Iso treat- ment, addition of the highest concentration of TFF-3 (1.5μM) appeared to slightly attenuate the ability of Iso to reduce Cch-induced pMLC expression. Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Figure 6: RV Stimulation Attenuates Formoterol (Form)-Induced Airway/HASM Relaxation (Left) hPCLS stimulated with RV and airway relaxation measured to form dose responses. n=10-18 distinct donors, *p < 0.05 (Right) HASM stimulated with media from control or RV exposed epithelial cells, then stim- ulated with form – cAMP production assessed by ELISA. n=7 distinct donors, *p < 0.05 Figure 7: TFF-3 Stimulation Attenuates Formoterol-In- duced Airway/HASM Relaxation (Left) hPCLS stimulated with TFF-3 and airway relaxa- tion measured to formoterol dose responses. n=4 dis- tinct donors, *p < 0.05 (Right) HASM stimulated TFF-3, then stimulated with formoterol – cAMP production as- sessed by ELISA. n=5 distinct donors, *p < 0.05 Figures 6 and 7 depict results from the PCLS assays, graphing the percentage of bronchodilation while comparing the control to treatments with either RV or TFF-3 at varying concentrations. Both RV-C15 and TFF-3 treatments of PCLS attenuated formoterol- induced airway relaxation. These figures also depict cAMP assay results, showing that both RV and TFF-3 exposure attenuates cAMP production by formoterol. Figure 8: cADDis assay to measure cAMP in live cells HASM cells received treatment with TFF-3 in varying concentrations, and cADDis assay measured the ef- fects in expression when exposed to formoterol and/or carbochol. In addition to the ELISA-based cAMP assays, we per- formed cADDis assays which measured the effect of TFF-3 treatment (24 hour pre-treatment) in the pres- ence and absence of Cch (20 µM, 15 minutes pre- treatment). TFF-3 pre-treatment appears to attenuate formoterol-induced cAMP production, which further decreases with co-stimulation from Cch (Figure 8). 4 DISCUSSION TFF-3 is known to for its ability to form trimers, which can enhance its role in signaling. Generally, it is rec- ognized for promoting epithelial cell health in the gas- trointestinal tract and assisting with epithelial repair during wound healing by reducing cell death through the activation of multiple receptors. However, it re- mains unclear whether TFF-3 affects the biology of air- way smooth muscle. Notably, TFF-3 release increased following stimulation of airway epithelial cells with RV- C15. Since TFF-3 appears to be associated with RV stimulation of the airways, its release may lead to acti- vation of one or more receptors. This potentially causes changes in the smooth muscle biology, result- ing in decreased cAMP production and reduced air- way relaxation. We used four different cell donor lines to analyze the Western blot results, providing a larger pool of data. The group mean fold change from the Western blot re- sults reveals the overall effects of phosphorylation of MLC. This serves as a readout for muscle contraction Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. following exposure to Cch and Iso reversing the phos- phorylation. The data shows that TFF-3 partially inhibits the Iso-dependent reversal of carbachol induced pMLC despite not augmenting carbachol-induced pMLC. It is possible that pMLC readout is not a sensitive enough to measure the effects of TFF-3 on airway smooth muscle relaxation. Hence, it is important to examine other tar- gets involved in relaxation going forward. We used precision cut lung slices (PCLS) to assess changes in formoterol-induced relaxation fol- lowing RV or TFF-3 exposure since formoterol is a mainstay bronchodilator used in asthma therapy. RV and TFF-3 was able to significantly reduce for- moterol’s effectiveness in relaxing the airways. Treat- ment with RV or TFF-3 at varying concentrations re- sulted in significantly less bronchodilation in response to formoterol compared to the control buffer stimula- tion. Future experiments are needed to determine whether RV-dependent attenuation of relaxation is TFF-3-dependent. Additionally, we used human airway smooth muscle to measure cAMP generation to indicate mus- cle relaxation. Results demonstrated that exposure of airway smooth muscle to either media from RV-stimu- lated epithelial cells or direct exposure to TFF-3 re- duced cAMP production by formoterol show a de- crease in the amount of cAMP generated by for- moterol exposure. This indicates that the relaxation of muscles following RV or TFF-3 stimulation decreases bronchodilation and relaxation responses. The cAD- Dis results show that TFF-3 stimulation decreases the responsiveness of airway smooth muscle to for- moterol, which the addition of Cch augments. These results suggest that TFF-3 may increase an inhibitory signal that Cch treatment enhances, however addi- tional confirmation would be needed. A major limitation of this study is the lack of understanding regarding how TFF-3 can elicit these types of responses. The specific mechanisms by which RV decreases responsiveness to bronchodilators and the receptors to which RV binds remain unclear. There are gaps in understanding how TFF-3, primarily recog- nized as a mediator in the gastrointestinal tract, modu- lates bronchodilation in HASM within the lungs. In future research related to TFF-3 and RV’s effects on asthmatic exacerbations, N numbers can be expanded to address response heterogeneity and further support these find- ings. Although animal models may be a valid choice for experimentation, observations reveal that mouse mod- els have not demonstrated TFF-3 expression or respon- siveness to bronchodilators in the same way that human airways do, which may limit the effectiveness of this model. 5 CONCLUSION In conclusion, the data identifies potential therapeutic targets that researchers can modulate to decrease the effects of RV on asthma exacerbations. Western blot results indicated that the highest con- centration of TFF-3 attenuated the ability of the Iso bronchodilator to reduce Cch-induced pMLC expres- sion. PCLS Assays visually demonstrate the tissue’s ability to attenuate cAMP production when treated with RV and TFF-3. The cADDis assay results show that TFF-3 stimulation reduces the airway’s responsiveness to bronchodilators, which in turn the addition of Cch can enhance. Gaining a better understanding of how TFF-3 signaling pathways alter bronchomotor tone will improve the efficacy of current treatments within the asthma therapeutic area. Our data suggests that TFF-3 may function as an inhibitory molecule that could serve as a novel therapeutic target to prevent bronchodilator hyporesponsiveness. These results of- fer hope for improving the current bronchodilators for those exposed to RV and for developing new thera- peutics to treat RV-dependent asthma exacerbations. Further studies would need to be conducted to deter- mine the exact mechanisms by which TFF-3 attenuates these responses. 6 REFERENCES [1] Bianchi, M. E., & Mezzapelle, R. (2020). The chemo- kine receptor CXCR4 in cell proliferation and tissue regeneration. Frontiers in Immunology, 11. https://doi.org/10.3389/fimmu.2020.02109 [2] Braga Emidio, N., Brierley, S. M., Schroeder, C. I., & Muttenthaler, M. (2020). 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International Journal of Molecular Sci- ences, 23(23), 15359. https://doi.org/10.3390/ijms232315359 https://doi.org/10.1097/md.0000000000034749 https://doi.org/10.1542/peds.2012-2183rrr https://doi.org/10.1038/s42003-020-01504-0 https://doi.org/10.1007/s11894-010-0131-2 https://doi.org/10.1007/s11894-010-0131-2 https://doi.org/10.1183/09031936.00186510 https://doi.org/10.3390/ijms232315359 Aresty Rutgers Undergraduate Research Journal, Volume I, Issue VI This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Mira Yin graduated in 2023 from Rutgers University New Bruns- wick School of Engineering and the Honors College with a B.S. in Biomedical Engineering with a minor in Biology. Mira worked as an undergraduate researcher in Dr. Cynthia Koziol-White’s lab within the Rutgers Institute for Translational Medicine and Science (RITMS) first through the Aresty Research Assistant program, and then continued her research post-program for an additional year. Her work focuses on understanding the signaling pathways tar- geted during viral infection, more specifically those that modulate airway tone. Outside of research, Mira was also a School of Engi- neering (SOE) Ambassador, President of the Habitat for Humanity organization, and Alumni Relations Chair for the Society of Women Engineers (SWE). Currently, Mira works at Merck & Co. within Global Clinical Trial Operations (GCTO), and is pursuing her M.S. in Clinical Research Management (CRM) in the Drug Safety and Pharmacovigilance track at Rutgers University. Mira can be contacted at mcy28@rutgers.edu. mailto:mcy28@rutgers.edu.