MIF family proteins in lung diseases
Song, Shanshan
DOI:
10.33612/diss.172449812
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Publication date: 2021
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Song, S. (2021). MIF family proteins in lung diseases. University of Groningen. https://doi.org/10.33612/diss.172449812
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GENERAL DISCUSSION AND
FUTURE PERSPECTIVES
The lung is of crucial importance to gas exchange and humans breathe in around 10,000 liters of air each day. Inhaled air contains many different airborne particles and microbes that can injure the lung. Maintenance of tissue homeostasis and repair following injury relies on the function of adult lung stem/progenitor cells. Studies have postulated that progenitor cells in the respiratory system are able to restart the cell cycle, proliferate and differentiate into one or more cell populations and thereby restore functionality of lung tissue (1–4). The work described in this thesis has investigated how MIF and D-DT could potentially affect differentiation and proliferation of adult epithelial progenitors.
As we discussed in chapter 2, tissue repair is an essential process that allows the replacement of damaged cells and the restoration of organ function, which is critical for survival. The adult lung is highly quiescent at steady state but has a remarkable healing capacity to quickly replenish epithelium and other cells after tissue injury. For example, in proximal conducting airways, club cells and basal cells are progenitors that have been shown to give rise to ciliated epithelial cells to maintain the bronchiolar epithelial structural and functional integrity. In distal alveoli, alveolar type 2 cells (AECII) have the capacity to give rise to alveolar type 1 cells (AECI) and can repair damaged alveoli (5). However, dysregulation of any of the cells or stages of wound healing or continuous repetitive injury could lead to pathological outcomes (6,7). For instance, continuous exposure to cigarette smoke is associated with the development of lung cancer, COPD, and pulmonary fibrosis. To date we have precious few possibilities to treat the damage done in these debilitating diseases. Understanding the underlying mechanisms of lung repair and being able to direct them to our advantage, will be an important contribution to the development of novel therapeutic strategies for damaged lungs.
The work in this thesis has shown that both MIF and D-DT are involved in COPD in the context of epithelial repair. As we reviewed in chapter 1, MIF is constitutively expressed and stored in intracellular vesicles. It is involved in inflammation and cell proliferation through five membrane receptors: CD74 and chemokine receptors CXCR2, CXCR4, ACKR3 and EGFR. MIF can also exert effects in the intracellular space directly. D-DT is a structural and functional homolog of MIF and also has biological effects through MIF’s receptor CD74.
To understand the roles of MIF and D-DT in lung diseases, we first investigated whether MIF and D-DT are produced in lung tissue. In chapters 3 and 4, we
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found that in healthy mouse lung tissue, MIF was predominantly present in ciliated epithelial cells and not in alveolar epithelial cells, while D-DT was mostly present in alveolar type 2 cells (AECII). In airways of control human lung tissue, both MIF and D-DT were detected in ciliated epithelial cells and D-DT expression was also seen in basal epithelial cells. In human alveoli, MIF was mainly expressed in macrophages while D-DT was shown in both macrophages and AECII (Fig. 1). Their different expression patterns in lung tissue already indicated that MIF and D-DT may have different functions in lung tissue.
▲Figure 1. Expression patterns of MIF and D-DT in nondiseased mouse and human lung
tissue. Representative pictures of lung tissue from a healthy mouse or human lung tissue from
a patient with normal lung function undergoing surgical resection for lung cancer stained for MIF or D-DT. A: In healthy mouse lung tissue, MIF and D-DT expression was observed in ciliated epithelial cells (black arrows) while D-DT expression was also observed in AECII (green arrows). B: In control human tissue, MIF and D-DT expression was observed in ciliated epithelial cells (black arrows) and macrophages (red arrows) while D-DT expression was also observed in basal cells (yellow arrows) and AECII (green arrows).
Expression of MIF family proteins and their receptors/interacting partners has been shown in most, if not all, tissues of the body and many studies have shown a role for these proteins in tissue injury (8–16). In chapter 1, we have summarized the evidence that clearly shows that MIF has detrimental and beneficial properties
in kidney, liver, brain and lung injury and participates in many processes in tissue injury. The divergent effects may be caused by the cellular context, timing and diverse physiological conditions as well as the known and unknown pathways in which MIF is involved. The evidence for a role of D-DT is limited, simply because it has not been studied much in any type of injury.
As we described in chapter 1, D-DT and MIF share striking similarities. D-DT has an overall structural fold similar to that of MIF, including the characteristic N-terminal proline (Pro-1), which is the key catalytic residue in the tautomerase activity of these proteins (17). CD74 is the common receptor for both MIF and D-DT, and as shown for MIF, D-DT has been reported to activate ERK signaling through CD74. Based on our data in chapter 3 and data from Chatterjee et al., both MIF and D-DT were found to promote cell survival through ACKR3-dependent Akt signalling (9). In addition, the genes encoding MIF and D-DT are both located on chromosome 22 (17,18). Therefore, it may seem that MIF and D-DT are redundant cytokines that function as backups for each other. Despite having similar overall structures, the amino acid sequences of D-DT and MIF only show 34% pairwise identity in humans and 27% in mice. Compared with MIF, D-DT lacks several important motifs that are essential binding sites. For example, D-DT does not have the pseudo-(E)LR (Arg11, Asp44) motif and RLR (Arg86-Leu87-Arg88) structural motif that gives MIF its chemokine-like function by being able to bind to CXCR2 and CXCR4, respectively (17,19,20). D-DT also lacks the CXXC motif that MIF has and which appears to be essential for redox activity (21). Together with the different expression pattern in lung tissue, this indicates that MIF and D-DT likely have different biological functions in lung tissue.
In chapter 3, we investigated the role of D-DT in lung tissue in more detail and we
showed that D-DT promoted proliferation of alveolar epithelial cells. Using a model of lung organoids, we found that D-DT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. In addition, we also demonstrated that D-DT contributed to this alveolar growth via ACKR3 to promote proliferation of AECII and/or protect them from apoptosis. In chapter 4, we then compared the effects of MIF and D-DT on lung epithelial progenitor cell function using the same murine lung organoid model. In contrast to D-DT stimulating proliferation of primary AECII, MIF surprisingly inhibited the regenerative ability of alveolar cells from control murine lung tissue. We further demonstrated that MIF impaired growth-support from fibroblasts as MIF pretreatment of fibroblasts impaired
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their ability to support outgrowth of alveolar structures. The critical cytokine for this effect may be TGFβ as expression of this factor by fibroblasts was induced in fibroblasts by MIF and TGFβ has previously been shown to inhibit growth of alveolar organoids (22). These data suggest that D-DT is not merely a backup protein for MIF, but clearly has its own functions, exemplified by its different expression pattern in the lung. It is therefore also essential to consider their intricate relationship for future therapeutic, diagnostic and prognostic use of MIF and D-DT.
Roles of MIF and D-DT in lung epithelial repair
in the context of COPD
Chronic obstructive pulmonary disease (COPD) is a common respiratory disease worldwide and is a major burden on health care with no curative treatments. Over the past decade, a lot of effort has gone into finding new therapies that target inflammation and oxidative stress in COPD, but with little success. An alternative and perhaps more promising approach may be to harness the endogenous lung repair capacity to restore lung function.
The data on MIF in COPD is confusing. Higher levels of MIF protein and mRNA were shown in serum, sputum, lung tissue, and lavage macrophages of COPD patients compared to healthy smokers and non-smoker controls(13,23,24). However, other studies have shown lower levels of MIF in serum of patients with severe disease compared to controls(25) and also lower plasma MIF levels in COPD patients compared to healthy smokers (11). These divergent results were also reported for animal models of COPD. Mice exposed to cigarette smoke for three months had higher MIF levels in bronchoalveolar lavage fluid whereas mice exposed for six months had lower MIF levels compared to air controls and this coincided with emphysema development (11). These data suggest that MIF is involved in some way in COPD but that its role may be complex and depend on situation/disease stage.
Less data is available for D-DT in COPD. We recently showed that D-DT mRNA levels were higher in lung tissue of patients with COPD compared to controls (24). A recent study of D-DT in the context of cigarette smoking found that D-DT-deficient mice were more susceptible to both spontaneous and cigarette smoke-induced emphysema compared to wild-type mice, similar to what was found for MIF-deficient mice (26), indicating that both proteins may have a role in lung repair.
In chapters 3 and 4, we therefore also investigated the effects of MIF and D-DT
on growth of lung epithelial cells derived from lung tissue of patients with different stages of disease severity using lung organoids. Similar to what we found with murine epithelial cells, D-DT treatment resulted in significantly more and larger alveolar organoids. This is good news as it indicates that D-DT treatment may also be able to stimulate lung repair in lungs affected by COPD. MIF, on the other hand, continued to emphasize its complicated role in lung tissue. MIF treatment of epithelial cells derived from lung tissue of control patients or patients with COPD GOLD stage I resulted in fewer organoids compared to non-treated cells. However, treating cells derived from lung tissue of patients with severe COPD with MIF resulted in more and bigger organoids compared to untreated control cells. These data not only highlight the divergent effects of MIF and D-DT on lung epithelial repair, but also the complicated role MIF has. D-DT contributes to alveolar repair via ACKR3. The divergent effects of MIF on lung epithelial repair emphasizes we still need to know more about the interacting partners of MIF in lung tissue and its pathological conditions.
Role of MIF and D-DT in lung cancer
According to the WHO, cancer is the second leading cause of death worldwide and lung cancer is the most deadly one with an estimated 2,09 million cases in 2018 (27). Conventional therapies for patients with lung cancer are available, including resection surgery, chemotherapy and radiotherapy (28). However, side effects of above conventional therapies significantly affect quality of life and are often not successful in eradicating the disease. In the past decades, the development of targeted therapy by small molecular compounds or antibodies, such as EGFR-tyrosine kinase inhibitors, seems to have at least reduced side effects (to some extent), but still 5-year survival chances are low (29,30). Therefore, identifying novel targets and developing new therapeutic combination strategies are still required.
Quite some studies have investigated MIF in the context of lung cancer, while the data for D-DT are again scarce. MIF levels were found to be significantly higher in serum and lung tissue from patients with non-small cell lung cancer than healthy individuals (31,32). In addition, MIF expression was found to correlated to disease stage with higher MIF levels in patients with advanced disease stage compared with those with early disease stage, suggesting MIF could be used as a biomarker of non-small cell lung cancer (33). The evidence of higher MIF levels and the correlation with poor
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prognosis in lung cancer patients demonstrates the importance of MIF in a clinical context but does not definitely prove that MIF may promote cancer development. With our work, however, we have added some pieces of evidence that MIF is important for lung cancer growth. We showed that degradation of intracellular MIF can inhibit the growth of lung cancer cells (Chapter 5). Importantly, MIF-directed PROTACs do not inhibit proliferation and differentiation of primary epithelial progenitor cells (Figure. 2). In addition, we also demonstrated that ACKR3, one of the receptors MIF can signal through, contributes to survival of non-small cell lung cancer cells (Chapter
6). Together with our novel finding in chapter 3 that D-DT can also interact with this
ACKR3 receptor, a similar role for D-DT may be envisaged.
Figure 2. The MIF-directed PROTAC MD13 doesn’t inhibit growth of murine alveolar organoids. (A) Quantification of alveolar, and airway organoid numbers (n=4) and (B) organoid
size at day 14 following treatment with/without different concentrations of MD13 (0.2 µM, 2 µM).
Our work also added some insight into how MIF may be promoting growth of lung cancer cells. As described in chapter 1, MIF has many extracellular and intracellular partners it can interact with for achieving biological effects associated with cancer growth such as promotion of cell growth and cell survival and inhibition of programmed cell death,
among others. In chapter 4, we showed that treating A549 lung cancer epithelial cells with recombinant human MIF did not affect cell proliferation. However, in chapter 5, treatment of these same A549 cells with a MIF-directed proteolysis targeting chimera (PROTAC) significantly inhibited their proliferation and growth of tumor spheroids. These data suggest that the role of intracellular MIF differs from that of extracellular (exogenously added) MIF, and that intracellular MIF may be more important in lung cancer.
A new tool for MIF study
Over the past two decades, many small-molecule MIF inhibitors with nanomolar potency have been developed. For instance, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) is a highly specific MIF tautomerase activity inhibitor and frequently used as the reference inhibitor for MIF (34). However, most MIF inhibitors bind within the hydrophobic pocket that contains the highly conserved amino acids known to be essential for MIF’s tautomerase activity and therefore they are only able to inhibit tautomerase activity. Although several studies demonstrated that some of these compounds can also inhibit the interaction between MIF and its main receptor CD74, the evidence that these MIF inhibitors work through inhibiting the interaction with CD74 and not through inhibiting the enzyme activity is often weak. Therefore, we developed a small-molecule protein degrader that can specifically induce the degradation of MIF by hijacking the ubiquitin-proteasome system (Chapter 5). This MIF degrader was designed by linking MIF-binding molecules to pomalidomide as a ligand for the cereblon Cullin RING E3 ubiquitin ligase complex, and this so-called PROTAC system induced almost complete MIF degradation in cancer cells. Therefore, it is a novel research tool that effectively induces MIF degradation, which helps us to further understand the biological function of intracellular MIF. Our work suggests that the use of MIF-directed PROTACs may be a potential therapeutic option for treating lung cancer.
Future Perspectives
Our work has implicated MIF and D-DT in the pathogenesis of lung diseases and both of them are important therapeutic targets in lung disease. However, considering the fact that MIF is a multifaceted protein interacting with many binding partners and participate in many processes, future therapeutic use of MIF agonists, inhibitors or degraders should take severity and stage of disease into consideration.
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In this thesis, we have shown that both MIF and D-DT treatments are beneficial for epithelial repair by promoting proliferation and differentiation of lung alveolar progenitor cells in severe COPD. However, MIF (and maybe D-DT) is also important for growth of lung cancer cells. Patients with COPD already have higher chance to develop lung cancer (35) and therefore treatment of COPD with MIF or D-DT may be a double-edged sword. Whether D-DT also stimulates lung cancer cell growth needs further study. Fortunately, the good news is that exogenous MIF treatment does not affect proliferation of lung cancer cells in vitro. It may suggest MIF treatment could be a safe therapy for severe COPD, although D-DT seems to be the better option, as it stimulates alveolar regeneration in all severity stages of COPD.
Interestingly, the use of a MIF-directed PROTAC significantly inhibited proliferation of A549 lung cancer cells, while exogenously added MIF inhibitors did not. These results indicate that intracellular MIF may be more important in lung cancer. However, exogenously added MIF did promote proliferation and differentiation of alveolar progenitor cells derived from lung tissue of patients with severe COPD. These data suggest that exogenously added MIF and intracellular MIF have effects on lung epithelial proliferation through different receptors under the microenvironment of CODP and lung cancer. For future study, there is thus an urgent need to elucidate the specific receptors that are involved in MIF-induced epithelial cell proliferation in COPD and lung cancer. This is important to unveil the precise therapeutic potential of MIF against COPD and lung cancer. Furthermore, small molecules with unique mechanisms of action on protein-protein binding inhibition are highly required. This thesis has shown novel roles of MIF and D-DT in lung epithelial repair that were not known before, although there is much we do not understand yet. It may help us design epithelial repair strategies for COPD that do not induce lung cancer and inhibition approaches for lung cancer cells that do not induce emphysema.
Declaration of my contributions
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