Although the CBAs HHA and GNA are not mitogenic and not toxic to mice when administered intravenously [113], caution must be taken in the development of the CBAs to use as antiviral drug in the clinic. First, the natural plant lectins are expensive to produce and hard to scale-up, but efforts have been made to express CBAs in commensal bacteria which provide an easy production process of this class of agents. Second, there can be a systemic reaction against the lectins such as in food allergies against peanut lectin or banana lectin [114,115]. Third, the CBAs can recognize aspecifically cellular glycans and could interfere with host cellular processes. But, DENV glycosylation is of the high-mannose or pauci-mannose type, which is only rare on mammalian proteins. The synthetic production of small non-peptidic molecules, such as PRM-S, with CBA-like activity, could overcome the pharmacological problems of the plant lectins. Therefore, PRM-S forms a potential lead

candidate in the development of more potent and specific DENV entry inhibitors.

In conclusion, besides active vector control in tropical and subtropical regions, there is an urgent need for antiviral treatment to protect half of the world's population against severe DENV infections. DC-SIGN is thought to be the most important DENV receptor and that the DC-SIGN-DENV envelope protein interaction is an excellent target for viral entry inhibitors such as the CBAs. Resistance against HHA forces the virus to delete its N-glycans and subsequently this mutant virus is not able anymore to infect its most important target cells.

a mosquito-derived virus, but this was not the case for DENV [101].

experiments of SA-17.

**5. Conclusion**

The glycosylation at Asn67 is demonstrated to be essential for infection of monocyte-derived dendritic cells (MDDC), indicating an interaction between DC-SIGN and the glycan at Asn67 [96,106]. Also the HHAres DENV was not able to infect efficiently DC-SIGN+ cells or cells that express the DC-SIGN-related liver-specific receptor L-SIGN. Interestingly, MDDC are also not susceptible for HHAres DENV infection, indicating the importance of the DC-SIGN-mediated DENV infection in MDDC. Moreover, cells of the hematopoietic origin, such as DC, are described to be necessary for DENV pathogenesis [84]. If the CBA resistant DENV in not able to infect DC anymore, it can be stated that the CBAs interfere with a physiologically highly relevant target. DC-SIGN is postulated as the most important DENV entry receptor until now. The entry process of DENV in Vero, Huh-7, BHK-21 and C6/36 cell lines is DC-SIGN-independent and also carbohydrate-independent. Indeed, HHAres DENV can efficiently enter and replicate in these cell lines. HHAres DENV lacking both N-glycans on the envelope E-glycoprotein is able to replicate efficiently in mammalian cells, with the exception of DC-SIGN+ cells.

The HHAres virus was used as a tool to identify the antiviral target of other classes of compounds as it could replicate in human liver Huh-7 cells. The use of Huh-7 cells has much more clinical relevance than using monkey (Vero) or hamster (BHK) kidney cells. The HHAres DENV was found cross-resistant to GNA, that recognizes like HHA, -1,3 mannose residues. UDA, which recognizes mainly the N-acetylglucosamine residues of the Nglycans, also lacked antiviral activity against HHAres DENV in Huh-7 cells. This indicates that the entire backbone of the N-glycan is deleted. Likewise, pradimicin-S (PRM-S), a smallsize -1,2-mannose-specific CBA, was also unable to inhibit HHAres DENV. This demonstrates that PRM-S targets also the N-glycans on the DENV envelope. In contrast, ribavirin (RBV), a nucleoside analogue and inhibitor of cellular purine synthesis [74], retained as expected wild-type antiviral activity against HHAres DENV. This argues against that there would be compensatory mutations in the non-structural proteins of DENV which are responsible for an overall enhanced replication of the viral genome [107,108]. SA-17, a novel doxorubicine analogue that inhibits the DENV entry process [109], was equipotent against WT and HHAres DENV. The SA-17 compound is predicted to interact with the hydrophobic binding pocket of the E-glycoprotein which is independent from the Nglycosylation state of the E-glycoprotein [109]. These data confirm the molecular docking experiments of SA-17.

Generally, the function of glycosylation on surface proteins is proper folding of the protein, trafficking in the endoplasmic reticulum, interaction with receptors and influencing virus immunogenicity [110]. Virions produced in the mosquito vector and human host may have structurally different N-linked glycans, because the glycosylation patterns are fundamentally different [101,111]. N-glycosylation in mammalian cells is often of the complex-type because a lot of different processing enzymes could add a diversity of monosaccharides. Glycans produced in insect cells are far less complex, because of less diversity in processing enzymes and usually contain more high-mannose and paucimannose-type glycans. DC-SIGN can distinguish between mosquito- and mammalian cellderived alphaviruses [112] and West Nile virus [68], resulting in a more efficient infection by a mosquito-derived virus, but this was not the case for DENV [101].

Although the CBAs HHA and GNA are not mitogenic and not toxic to mice when administered intravenously [113], caution must be taken in the development of the CBAs to use as antiviral drug in the clinic. First, the natural plant lectins are expensive to produce and hard to scale-up, but efforts have been made to express CBAs in commensal bacteria which provide an easy production process of this class of agents. Second, there can be a systemic reaction against the lectins such as in food allergies against peanut lectin or banana lectin [114,115]. Third, the CBAs can recognize aspecifically cellular glycans and could interfere with host cellular processes. But, DENV glycosylation is of the high-mannose or pauci-mannose type, which is only rare on mammalian proteins. The synthetic production of small non-peptidic molecules, such as PRM-S, with CBA-like activity, could overcome the pharmacological problems of the plant lectins. Therefore, PRM-S forms a potential lead candidate in the development of more potent and specific DENV entry inhibitors.

## **5. Conclusion**

176 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

to predict virus phenotypes.

exception of DC-SIGN+ cells.

N-glycosylation sites, had a similar plaque phenotype in BHK cells (manuscript submitted). It has been shown that DENV lacking the glycosylation at Asn67 resulted in a replicationdefective phenotype, because this virus infects mammalian cells weakly and there is a reduced secretion of DENV E-protein. Replication in mosquito cells was not affected, because the mosquito cells restore the N-glycosylation at Asn67 with a compensatory sitemutation (K64N) generating a new glycosylation site [96,105]. These data are in contrast with other published results, where was demonstrated that DENV lacking the Asn67-linked glycosylation can grow efficiently in mammalian cells, depending on the viral strain and the amino acid substitution abolishing the glycosylation process [102]. A compensatory mutation was detected (N124S) to repair the growth defect without creating a new glycosylation site. Thus, the glycan at Asn67 is not necessary for virus growth, but a critical role for this glycan in virion release from mosquito cells was demonstrated [102]. However, HHA resistant virus was found to replicate efficiently in mosquito and insect cells indicating an efficient carbohydrate-independent viral replication in these cell lines. A possible explanation for the differences between our data and data from previous studies could be that the mutant virus has been generated in mosquito C6/36 cells (during replication under antiviral drug pressure) and not in mammalian cells (after introducing the mutations by site-directed mutagenesis). In addition, in previous studies, other amino acid substitutions were generated, resulting in different virus genotypes and subsequently resulting in poorly

The glycosylation at Asn67 is demonstrated to be essential for infection of monocyte-derived dendritic cells (MDDC), indicating an interaction between DC-SIGN and the glycan at Asn67 [96,106]. Also the HHAres DENV was not able to infect efficiently DC-SIGN+ cells or cells that express the DC-SIGN-related liver-specific receptor L-SIGN. Interestingly, MDDC are also not susceptible for HHAres DENV infection, indicating the importance of the DC-SIGN-mediated DENV infection in MDDC. Moreover, cells of the hematopoietic origin, such as DC, are described to be necessary for DENV pathogenesis [84]. If the CBA resistant DENV in not able to infect DC anymore, it can be stated that the CBAs interfere with a physiologically highly relevant target. DC-SIGN is postulated as the most important DENV entry receptor until now. The entry process of DENV in Vero, Huh-7, BHK-21 and C6/36 cell lines is DC-SIGN-independent and also carbohydrate-independent. Indeed, HHAres DENV can efficiently enter and replicate in these cell lines. HHAres DENV lacking both N-glycans on the envelope E-glycoprotein is able to replicate efficiently in mammalian cells, with the

The HHAres virus was used as a tool to identify the antiviral target of other classes of compounds as it could replicate in human liver Huh-7 cells. The use of Huh-7 cells has much more clinical relevance than using monkey (Vero) or hamster (BHK) kidney cells. The HHAres DENV was found cross-resistant to GNA, that recognizes like HHA, -1,3 mannose residues. UDA, which recognizes mainly the N-acetylglucosamine residues of the Nglycans, also lacked antiviral activity against HHAres DENV in Huh-7 cells. This indicates that the entire backbone of the N-glycan is deleted. Likewise, pradimicin-S (PRM-S), a smallsize -1,2-mannose-specific CBA, was also unable to inhibit HHAres DENV. This

In conclusion, besides active vector control in tropical and subtropical regions, there is an urgent need for antiviral treatment to protect half of the world's population against severe DENV infections. DC-SIGN is thought to be the most important DENV receptor and that the DC-SIGN-DENV envelope protein interaction is an excellent target for viral entry inhibitors such as the CBAs. Resistance against HHA forces the virus to delete its N-glycans and subsequently this mutant virus is not able anymore to infect its most important target cells.

Thus the CBAs act in two different ways: prevention of viral entry by directly binding Nglycans on the viral envelope and indirectly forcing the virus to delete its N-glycans and loose the capability to infect DC. The plant lectins provided more insight into the entry pathway of the virus into the host cell. Hopefully some of these future derivatives with a comparable mode of action will reach clinical trials in the near future.

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection 179

[10] Mairuhu ATA, Wagenaar J, Brandjes DPM, van Gorp ECM (2004) Dengue: an arthropod-borne disease of global importance. Eur J Clin Microbiol Infect Dis 23: 425-

[11] Rigau-Pérez JG, Clark GG, Gubler DJ, Reiter P, Sanders RJ, Vorndam AV (1998)

[12] Solomon T, Mallewa M (2001) Dengue and other emerging flaviviruses. J Infect 42:

[13] Halstead SB (1988) Pathogenesis of dengue: challenges to molecular biology. Science

[14] Takada A, Kawaoka Y (2003) Antibody-dependent enhancement of viral infection:

[15] Kliks SC, Nimmanitya S, Nisalak A, Burke DS (1988) Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants.

[16] Halstead SB (1979) In vivo enhancement of dengue virus infection in rhesus monkeys

[17] Wu SJL, Grouard-Vogel G, Sun W, Mascola JR, Brachtel E, Putvatana R et al. (2000) Human skin Langerhans cells are targets of dengue virus infection. Nat Med 6: 816-820. [18] Kou Z, Quinn M, Chen HY, Rodrigo WW, Rose RC, Schlesinger JJ et al. (2008) Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells. J Med Virol 80: 134-146. [19] Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E et al. (2002) Structure of dengue virus: Implications for flavivirus organization, maturation, and

[20] Acosta EG, Castilla V, Damonte EB (2008) Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrin-mediated endocytosis. J Gen Virol

[21] van der Schaar HM, Rust MJ, Chen C, van der Ende-Metselaar H, Wilschut J, Zhuang XW et al. (2008) Dissecting the cell entry pathway of dengue virus by single-particle

[22] Chu JJH, Ng ML (2004) Infectious entry of West Nile virus occurs through a clathrin-

[23] Nawa M, Takasaki T, Yamada K, Kurane I, Akatsuka T (2003) Interference in Japanese encephalitis virus infection of vero cells by a cationic amphiphilic drug,

[24] Acosta EG, Castilla V, Damonte EB (2009) Alternative infectious entry pathways for

[25] Modis Y, Ogata S, Clements D, Harrison SC (2004) Structure of the dengue virus

[26] Stiasny K, Fritz R, Pangerl K, Heinz FX (2009) Molecular mechanisms of flavivirus

dengue virus serotypes into mammalian cells. Cell Microbiol 11: 1533-1549.

envelope protein after membrane fusion. Nature 427: 313-319.

molecular mechanisms and in vivo implications. Rev Med Virol 13: 387-398.

Dengue and dengue haemorrhagic fever. Lancet 352: 971-977.

by passively transferred antibody. J Infect Dis 140: 527-533.

tracking in living cells. Plos Pathog 4: e1000244.

chlorpromazine. J Gen Virol 84: 1737-1741.

membrane fusion. Amino Acids 41: 1159-1163.

mediated endocytic pathway. J Virol 78: 10543-10555.

433.

104-115.

239: 476-481.

Am J Trop Med Hyg 38: 411-419.

fusion. Cell 108: 717-725.

89: 474-484.

## **Author details**

Dominique Schols\* and Marijke Alen *Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium*

## **Acknowledgement**

We are grateful to S. Claes, B. Provinciael, E. Van Kerckhove and E. Fonteyn for excellent technical assistance. This work was supported by the FWO (G-485-08N and G.0528.12N), the KU Leuven (PF/10/018 and GOA/10/014) and the Dormeur Investment Service Ltd.

## **6. References**

<sup>\*</sup> Corresponding Author

[10] Mairuhu ATA, Wagenaar J, Brandjes DPM, van Gorp ECM (2004) Dengue: an arthropod-borne disease of global importance. Eur J Clin Microbiol Infect Dis 23: 425- 433.

178 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

and Marijke Alen

dengue virus. Infect Genet Evol 3: 19-28.

[5] Halstead SB (2007) Dengue. Lancet 370: 1644-1652.

 Nat Rev Microbiol 5: 518-528.

dengue transmission. Nature 476: 454-457.

populations. Nature 476: 450-453.

**Author details**

Dominique Schols\*

**6. References**

S109.

291.

 \*

Corresponding Author

**Acknowledgement**

*University of Leuven, Leuven, Belgium*

comparable mode of action will reach clinical trials in the near future.

*Department of Microbiology and Immunology, Rega Institute for Medical Research,*

Thus the CBAs act in two different ways: prevention of viral entry by directly binding Nglycans on the viral envelope and indirectly forcing the virus to delete its N-glycans and loose the capability to infect DC. The plant lectins provided more insight into the entry pathway of the virus into the host cell. Hopefully some of these future derivatives with a

We are grateful to S. Claes, B. Provinciael, E. Van Kerckhove and E. Fonteyn for excellent technical assistance. This work was supported by the FWO (G-485-08N and G.0528.12N), the

[1] Holmes EC, Twiddy SS (2003) The origin, emergence and evolutionary genetics of

[2] Mackenzie JS, Gubler DJ, Petersen LR (2004) Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 10: S98-

[3] Halstead SB (2008) Dengue virus - Mosquito interactions. Annu Rev Entomol 53: 273-

[4] Gubler DJ (1998) Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480-496.

[6] Monath TP (1994) Dengue - the risk to developed and developing countries. Proc Natl

[7] Whitehead SS, Blaney JE, Durbin AP, Murphy BR (2007) Prospects for a dengue virus

[8] Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, McMeniman CJ et al. (2011) The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti

[9] Hoffmann AA, Montgomery BL, Popovici J, Iturbe-Ormaetxe I, Johnson PH, Muzzi F et al. (2011) Successful establishment of Wolbachia in Aedes populations to suppress

KU Leuven (PF/10/018 and GOA/10/014) and the Dormeur Investment Service Ltd.

	- [27] Martin CSS, Liu CY, Kielian M (2009) Dealing with low pH: entry and exit of alphaviruses and flaviviruses. Trends Microbiol 17: 514-521.

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection 181

[42] Alen MMF, Kaptein SJF, De Burghgraeve T, Balzarini J, Neyts J, Schols D (2009) Antiviral activity of carbohydrate-binding agents and the role of DC-SIGN in dengue

[43] Lozach PY, Burleigh L, Staropoli I, Navarro-Sanchez E, Harriague J, Virelizier JL et al. (2005) Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-

[44] Navarro-Sanchez E, Altmeyer R, Amara A, Schwartz O, Fieschi F, Virelizier JL et al. (2003) Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses.

[45] Tassaneetrithep B, Burgess TH, Granelli-Piperno A, Trumpfherer C, Finke J, Sun W et al. (2003) DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J

[46] van Kooyk Y, Geijtenbeek TBH (2003) DC-SIGN: Escape mechanism for pathogens.

[47] Mitchell DA, Fadden AJ, Drickamer K (2001) A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR - Subunit organization and

[48] Appelmelk BJ, van Die I, van Vliet SJ, Vandenbroucke-Grauls CMJE, Geijtenbeek TBH, van Kooyk Y (2003) Cutting edge: Carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells. J

[49] Feinberg H, Mitchell DA, Drickamer K, Weis WI (2001) Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294: 2163-2166. [50] Geijtenbeek TBH, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GCF, Middel J et al. (2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances

[51] Pöhlmann S, Zhang J, Baribaud F, Chen ZW, Leslie G, Lin G et al. (2003) Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol 77: 4070-4080. [52] Marzi A, Möller P, Hanna SL, Harrer T, Eisemann J, Steinkasserer A et al. (2007) Analysis of the interaction of Ebola virus glycoprotein with DC-SIGN (dendritic cellspecific intercellular adhesion molecule 3-grabbing nonintegrin) and its homologue DC-

[53] Geijtenbeek TBH, Krooshoop DJEB, Bleijs DA, van Vliet SJ, van Duijnhoven GCF, Grabovsky V et al. (2000) DC-SIGN-ICAM-2 interaction mediates dendritic cell

[54] Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature

SIGN internalization signals. J Biol Chem 280: 23698-23708.

binding to multivalent ligands. J Biol Chem 276: 28939-28945.

virus infection. Virology 387: 67-75.

Embo Rep 4: 723-728.

Exp Med 197: 823-829.

Nat Rev Immunol 3: 697-709.

Immunol 170: 1635-1639.

trans-infection of T cells. Cell 100: 587-597.

SIGNR. J Infect Dis 196 (Suppl 2): S237-S246.

trafficking. Nat Immunol 1: 353-357.

392: 245-252.

[42] Alen MMF, Kaptein SJF, De Burghgraeve T, Balzarini J, Neyts J, Schols D (2009) Antiviral activity of carbohydrate-binding agents and the role of DC-SIGN in dengue virus infection. Virology 387: 67-75.

180 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

for antiviral drug design. Antiviral Res 80: 11-22.

Virol 75: 7769-7773.

Exp Med 146: 218-229.

J Immunol 166: 1499-1506.

serotypes. J Gen Virol 90: 799-809.

to classic dengue fever. Virology 376: 429-435.

hemorrhagic-fever. Am J Trop Med Hyg 40: 444-451.

alphaviruses and flaviviruses. Trends Microbiol 17: 514-521.

[27] Martin CSS, Liu CY, Kielian M (2009) Dealing with low pH: entry and exit of

[28] Zhang Y, Zhang W, Ogata S, Clements D, Strauss JH, Baker TS et al. (2004) Conformational changes of the flavivirus E glycoprotein. Structure 12: 1607-1618. [29] Perera R, Khaliq M, Kuhn RJ (2008) Closing the door on flaviviruses: Entry as a target

[30] Modis Y, Ogata S, Clements D, Harrison SC (2003) A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A 100: 6986-6991. [31] Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC (1995) The envelope glycoprotein from tick-borne encephalitis virus at 2 angstrom resolution. Nature 375: 291-298. [32] Crill WD, Roehrig JT (2001) Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J

[33] Rajamanonmani R, Nkenfou C, Clancy P, Yau YH, Shochat SG, Sukupolvi-Petty S et al. (2009) On a mouse monoclonal antibody that neutralizes all four dengue virus

[34] Scherret JH, Mackenzie JS, Khromykh AA, Hall RA (2001) Biological significance of glycosylation of the envelope protein of Kunjin virus. Ann N Y Acad Sci 951: 361-363. [35] Halstead SB, O'rourke EJ, Allison AC (1977) Dengue viruses and mononuclear phagocytes .II. Identity of blood and tissue leukocytes supporting in vitro infection. J

[36] Durbin AP, Vargas MJ, Wanionek K, Hammond SN, Gordon A, Rocha C et al. (2008) Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared

[37] Kliks SC, Nisalak A, Brandt WE, Wahl L, Burke DS (1989) Antibody-dependent enhancement of dengue virus growth in human-monocytes as a risk factor for dengue

[38] Taweechaisupapong S, Sriurairatana S, Angsubhakorn S, Yoksan S, Khin MM, Sahaphong S et al. (1996) Langerhans cell density and serological changes following intradermal immunisation of mice with dengue 2 virus. J Med Microbiol 45: 138-145. [39] Ho LJ, Wang JJ, Shaio MF, Kao CL, Chang DM, Han SW et al. (2001) Infection of human dendritic cells by Dengue virus causes cell maturation and cytokine production.

[40] Libraty DH, Pichyangkul S, Ajariyakhajorn C, Endy TP, Ennis FA (2001) Human dendritic cells are activated by dengue virus infection: Enhancement by gamma

[41] Marovich M, Grouard-Vogel G, Louder M, Eller M, Sun W, Wu SJ et al. (2001) Human dendritic cells as targets of dengue virus infection. J Invest Dermatol 6: 219-224.

interferon and implications for disease pathogenesis. J Virol 75: 3501-3508.

	- [55] Nair MPN, Reynolds JL, Mahajan SD, Schwartz SA, Aalinkeel R, Bindukumar B et al. (2005) RNAi-directed inhibition of DC-SIGN by dendritic cells: Prospects for HIV-1 therapy. AAPS J 7: E572-E578.

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection 183

[68] Davis CW, Nguyen HY, Hanna SL, Sanchez MD, Doms RW, Pierson TC (2006) West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and

[69] Zellweger RM, Prestwood TR, Shresta S (2010) Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell

[70] Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M (1998) Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and

[71] Seneviratne SL, Malavige GN, de Silva HJ (2006) Pathogenesis of liver involvement

[72] Murrell S, Wu SC, Butler M (2011) Review of dengue virus and the development of a

[73] Murphy BR, Whitehead SS (2011) Immune response to dengue virus and prospects for

[74] Leyssen P, De Clercq E, Neyts J (2008) Molecular strategies to inhibit the replication of

[75] Sampath A, Padmanabhan R (2009) Molecular targets for flavivirus drug discovery.

[76] Balzarini J (2006) Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral

[77] François KO, Balzarini J (2010) Potential of carbohydrate-binding agents as

[78] Balzarini J, Van Herrewege Y, Vermeire K, Vanham G, Schols D (2007) Carbohydratebinding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-directed HIV-1 transmission to T

[79] Bertaux C, Daelemans D, Meertens L, Cormier EG, Reinus JF, Peumans WJ et al. (2007) Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited

[80] Balzarini J, Schols D, Neyts J, Van Damme E, Peumans W, De Clercq E (1991) Alpha- (1-3)-D-mannose-specific and alpha-(1-6)-D-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro.

[81] Harata K, Muraki M (2000) Crystal structures of Urtica dioica agglutinin and its complex with tri-N-acetylchitotriose. Journal of Molecular Biology 297: 673-681. [82] Hung SL, Lee PL, Chen HW, Chen LK, Kao CL, King CC (1999) Analysis of the steps

[83] Grassi F, Dezutter-Dambuyant C, McIlroy D, Jacquet C, Yoneda K, Imamura S et al. (1998) Monocyte-derived dendritic cells have a phenotype comparable to that of dermal

by carbohydrate-binding agents but not by polyanions. Virology 366: 40-50.

involved in dengue virus entry into host cells. Virology 257: 156-167.

during dengue viral infections. Trans R Soc Trop Med Hyg 100: 608-614.

infection. J Virol 80: 1290-1301.

apoptosis. J Immunol 161: 6338-6346.

vaccine. Biotechnol Adv 29: 239-247.

RNA viruses. Antiviral Res 78: 9-25.

lymphocytes. Mol Pharmacol 71: 3-11.

Antimicrob Agents Chemother 35: 410-416.

Antiviral Res 81: 6-15.

Res 71: 237-247.

a vaccine. Annu Rev Immunol 29: 587-619.

therapeutics against enveloped viruses. Med Res Rev .

Host Microbe 7: 128-139.

[68] Davis CW, Nguyen HY, Hanna SL, Sanchez MD, Doms RW, Pierson TC (2006) West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection. J Virol 80: 1290-1301.

182 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

new class of anti-infectives. Curr Med Chem 19: 992-1007.

virus in monocyte-derived dendritic cells. Plos One 6: e21658.

therapy. AAPS J 7: E572-E578.

Virol 76: 9877-9887.

S A 98: 2670-2675.

Pathog 4: e17.

cell responses. J Immunol 176: 426-440.

virus infection. J Virol 79: 7291-7299.

[55] Nair MPN, Reynolds JL, Mahajan SD, Schwartz SA, Aalinkeel R, Bindukumar B et al. (2005) RNAi-directed inhibition of DC-SIGN by dendritic cells: Prospects for HIV-1

[56] Dakappagari N, Maruyama T, Renshaw M, Tacken P, Figdor C, Torensma R et al. (2006) Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T

[57] Anderluh M, Jug G, Svajger U, Obermajer N (2012) DC-SIGN antagonists, a potential

[58] Chen YC, Wang SY (2002) Activation of terminally differentiated human monocytes/macrophages by dengue virus: Productive infection, hierarchical production of innate cytokines and chemokines, and the synergistic effect of lipopolysaccharide. J

[59] Miller JL, Dewet BJM, Martinez-Pomares L, Radcliffe CM, Dwek RA, Rudd PM et al. (2008) The mannose receptor mediates dengue virus infection of macrophages. Plos

[60] Kwan WH, Helt AM, Maranon C, Barbaroux JB, Hosmalin A, Harris E et al. (2005) Dendritic cell precursors are permissive to dengue virus and human immunodeficiency

[61] Alen MMF, De Burghgraeve T, Kaptein SJF, Balzarini J, Neyts J, Schols D (2011) Broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue

[62] de Witte L, Bobardt M, Chatterji U, Degeest G, David G, Geijtenbeek TB et al. (2007) Syndecan-3 is a dendritic cell-specific attachment receptor for HIV-1. Proc Natl Acad Sci

[63] Lambert AA, Gilbert C, Richard M, Beaulieu AD, Tremblay MJ (2008) The C-type lectin surface receptor DICIR acts as a new attachment factor for HIV-1 in dendritic cells

[64] Chen ST, Lin YL, Huang MT, Wu MF, Cheng SC, Lei HY et al. (2008) CLEC5A is

[65] Watson AA, Lebedev AA, Hall BA, Fenton-May AE, Vagin AA, Dejnirattisai W et al. (2011) Structural flexibility of the macrophage dengue virus receptor CLEC5A

[66] Bashirova AA, Geijtenbeek TBH, van Duijnhoven GCF, van Vliet SJ, Eilering JBG, Martin MP et al. (2001) A dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J Exp Med 193: 671-678. [67] Pöhlmann S, Soilleux EJ, Baribaud F, Leslie GJ, Morris LS, Trowsdale J et al. (2001) DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc Natl Acad Sci U

and contributes to trans- and cis-infection pathways. Blood 112: 1299-1307.

implications for ligand binding and signaling. J Biol Chem 286: 24208-24218.

critical for dengue-virus-induced lethal disease. Nature 453: 672-676.

dendritic cells and display ultrastructural granules distinct from Birbeck granules. J Leukoc Biol 64: 484-493.

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection 185

[98] Pugachev KV, Guirakhoo F, Ocran SW, Mitchell F, Parsons M, Penal C et al. (2004)

[99] Thai KTD, Henn MR, Zody MC, Tricou V, Nguyet NM, Charlebois P et al. (2012) High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1

[100] Johnson AJ, Guirakhoo F, Roehrig JT (1994) The envelope glycoproteins of dengue-1 and dengue-2 viruses grown in mosquito cells differ in their utilization of potential

[101] Hacker K, White L, de Silva AM (2009) N-Linked glycans on dengue viruses grown in

[102] Lee E, Leang SK, Davidson A, Lobigs M (2010) Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release. J Virol 84: 5171-5180. [103] Guirakhoo F, Hunt AR, Lewis JG, Roehrig JT (1993) Selection and partial characterization of dengue-2 virus mutants that induce fusion at elevated pH. Virology

[104] Lee E, Weir RC, Dalgarno L (1997) Changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the

[105] Bryant JE, Calvert AE, Mesesan K, Crabtree MB, Volpe KE, Silengo S et al. (2007) Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes. Virology

[106] Pokidysheva E, Zhang Y, Battisti AJ, Bator-Kelly CM, Chipman PR, Xiao CA et al. (2006) Cryo-EM reconstruction of dengue virus in complex with the carbohydrate

[107] Wang QY, Bushell S, Qing M, Xu HY, Bonavia A, Nunes S et al. (2011) Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J Virol 85: 6548-

[108] Xie X, Wang QY, Xu HY, Qing M, Kramer L, Yuan Z et al. (2011) Inhibition of dengue

[109] Kaptein SJF, De Burghgraeve T, Froeyen M, Pastorino B, Alen MMF, Mondotte JA et al. (2010) A derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro. Antimicrob Agents Chemother 54: 5269-5280. [110] Vigerust DJ, Shepherd VL (2007) Virus glycosylation: role in virulence and immune

[111] Rogers KM, Heise M (2009) Modulation of cellular tropism and innate antiviral

[112] Klimstra WB, Nangle EM, Smith MS, Yurochko AD, Ryman KD (2003) DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between

mosquito cell- and mammalian cell-derived viruses. J Virol 77: 12022-12032.

High fidelity of yellow fever virus RNA polymerase. J Virol 78: 1032-1038.

infection identifies mixed infections. J Virol 86: 835-843.

mammalian and insect cells. J Gen Virol 90: 2097-2106.

recognition domain of DC-SIGN. Cell 124: 485-493.

interactions. Trends Microbiol 15: 211-218.

response by viral glycans. J Innate Immun 1: 405-412.

virus by targeting viral NS4B protein. J Virol 85: 11183-11195.

glycosylation sites. Virology 203: 241-249.

194: 219-223.

366: 415-423.

6556.

protein. Virology 232: 281-290.

[98] Pugachev KV, Guirakhoo F, Ocran SW, Mitchell F, Parsons M, Penal C et al. (2004) High fidelity of yellow fever virus RNA polymerase. J Virol 78: 1032-1038.

184 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

Leukoc Biol 64: 484-493.

J Immunol 166: 1499-1506.

Agents Chemother 54: 1425-1435.

Agents Chemother 54: 3287-3301.

Virol 81: 7136-7148.

dendritic cells and display ultrastructural granules distinct from Birbeck granules. J

[84] Pham AM, Langlois RA, TenOever BR (2012) Replication in cells of hematopoietic

[85] Relloso M, Puig-Kröger A, Pello OM, Rodriguez-Fernandez JL, de la Rosa G, Longo N et al. (2002) DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated

[86] Tacken PJ, de Vries IJM, Torensma R, Figdor CG (2007) Dendritic-cell immunotherapy:

[87] Ho LJ, Wang JJ, Shaio MF, Kao CL, Chang DM, Han SW et al. (2001) Infection of human dendritic cells by Dengue virus causes cell maturation and cytokine production.

[88] Sun P, Fernandez S, Marovich MA, Palmer DR, Celluzzi CM, Boonnak K et al. (2009) Functional characterization of ex vivo blood myeloid and plasmacytoid dendritic cells

[89] Dejnirattisai W, Duangchinda T, Lin CLS, Vasanawathana S, Jones M, Jacobs M et al. (2008) A complex interplay among virus, dendritic cells, T cells, and cytokines in

[90] Palmer DR, Sun PF, Celluzzi C, Bisbing J, Pang SN, Sun W et al. (2005) Differential effects of dengue virus on infected and bystander dendritic cells. J Virol 79: 2432-2439. [91] Balzarini J, François KO, Van Laethem K, Hoorelbeke B, Renders M, Auwerx J et al. (2010) Pradimicin S, a highly soluble nonpeptidic small-size carbohydrate-binding antibiotic, is an anti-HIV drug lead for both microbicidal and systemic use. Antimicrob

[92] Hoorelbeke B, Huskens D, Férir G, François KO, Takahashi A, Van Laethem K et al. (2010) Actinohivin, a broadly neutralizing prokaryotic lectin, inhibits HIV-1 infection by specifically targeting high-mannose-type glycans on the gp120 envelope. Antimicrob

[93] Alexandre KB, Gray ES, Mufhandu H, McMahon JB, Chakauya E, O'Keefe BR et al. (2012) The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the

[94] Huskens D, Férir G, Vermeire K, Kehr JC, Balzarini J, Dittmann E et al. (2010) Microvirin, a novel alpha(1,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-HIV-1 activity comparable with that of cyanovirin-N but a much

[95] Swanson MD, Winter HC, Goldstein IJ, Markovitz DM (2010) A lectin isolated from

[96] Mondotte JA, Lozach PY, Amara A, Gamarnik AV (2007) Essential role of dengue virus envelope protein N-glycosylation at asparagine-67 during viral propagation. J

bananas is a potent inhibitor of HIV replication. J Biol Chem 285: 8646-8655.

[97] Ploegh H, Neefjes JJ (1990) Protein glycosylation. Curr Opin Cell Biol 2: 1125-1130.

DC-SIGN receptor and transfer to CD4(+) cells. Virology 423: 175-186.

origin is necessary for Dengue virus dissemination. Plos Pathog 8: e1002465.

by IFN, TGF-beta, and anti-inflammatory agents. J Immunol 168: 2634-2643.

from ex vivo loading to in vivo targeting. Nat Rev Immunol 7: 790-802.

after infection with dengue virus. Virology 383: 207-215.

Dengue virus infections. J Immunol 181: 5865-5874.

higher safety profile. J Biol Chem 285: 24845-24854.

	- [113] Balzarini J, Hatse S, Vermeire K, Princen K, Aquaro S, Perno CF et al. (2004) Mannosespecific plant lectins from the Amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection. Antimicrob Agents Chemother 48: 3858-3870.

**Chapter 9**

© 2012 Dorscheid et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Dorscheid et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**IL-13, Asthma and Glycosylation**

Samuel J. Wadsworth, S. Jasemine Yang and Delbert R. Dorscheid

Pulmonary function tests (PFTs/spirometry) are routinely used clinically to diagnose asthma. Forced expiratory volume in one second (FEV1) is a measure of airflow and is influenced largely by the resistance in the airways. During an "asthma attack", the airways narrow owing to mucus hypersecretion and smooth muscle contraction, which results in a sharp increase in airway resistance and reduction in FEV1. In the clinics, this can be reproduced by exposing patients with asthma to non-specific smooth muscle contractile agonists such as methacholine. A fall in FEV1 at a relatively low dose of methacholine indicates airway hyperresponsiveness (AHR). In asthmatic patients this drop in FEV1 can be reversed by inhaled bronchodilators such as β2-agonists (e.g. salbutamol), which relaxes airway smooth muscles. AHR is often used as a diagnostic

Asthma is increasingly considered a syndrome, with diverse overlapping pathologies and phenotypes contributing to significant heterogeneity in clinical manifestation, disease progression, and treatment response [1]. Severe uncontrolled asthmatics make up approximately 5-10% of the population of asthmatics, yet they consume around 50% of the treatment resources [2]. Spirometric tests are incapable of exposing the underlying pathologies or phenotypes which combine within an individual to produce the asthmatic disease. In contrast specific biomarkers may enable more accurate sub-phenotyping of disease by indicating the pathology within an individual, and assist clinicians to better tailor the type and/or dose of therapy. In this way biomarkers have the potential to guide more effective personalized treatment regimes

**in Airway Epithelial Repair**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51970

**1.1. Clinical assessment of asthma**

**1. Introduction**

criterion for asthma.

in asthma.

**Chapter 9**

## **IL-13, Asthma and Glycosylation in Airway Epithelial Repair**

Samuel J. Wadsworth, S. Jasemine Yang and Delbert R. Dorscheid

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51970

## **1. Introduction**

186 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

class I chitinases. Clin Exp Allergy 29: 673-680.

Chemother 48: 3858-3870.

[113] Balzarini J, Hatse S, Vermeire K, Princen K, Aquaro S, Perno CF et al. (2004) Mannosespecific plant lectins from the Amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection. Antimicrob Agents

[114] Rougé P, Culerrier R, Granier C, Rancé F, Barre A (2010) Characterization of IgEbinding epitopes of peanut (Arachis hypogaea) PNA lectin allergen cross-reacting with

other structurally related legume lectins. Molecular Immunology 47: 2359-2366. [115] Sanchez-Monge R, Blanco C, Diaz-Perales A, Collada C, Carrillo T, Aragoncillo C et al. (1999) Isolation and characterization of major banana allergens: identification as fruit

## **1.1. Clinical assessment of asthma**

Pulmonary function tests (PFTs/spirometry) are routinely used clinically to diagnose asthma. Forced expiratory volume in one second (FEV1) is a measure of airflow and is influenced largely by the resistance in the airways. During an "asthma attack", the airways narrow owing to mucus hypersecretion and smooth muscle contraction, which results in a sharp increase in airway resistance and reduction in FEV1. In the clinics, this can be reproduced by exposing patients with asthma to non-specific smooth muscle contractile agonists such as methacholine. A fall in FEV1 at a relatively low dose of methacholine indicates airway hyperresponsiveness (AHR). In asthmatic patients this drop in FEV1 can be reversed by inhaled bronchodilators such as β2-agonists (e.g. salbutamol), which relaxes airway smooth muscles. AHR is often used as a diagnostic criterion for asthma.

Asthma is increasingly considered a syndrome, with diverse overlapping pathologies and phenotypes contributing to significant heterogeneity in clinical manifestation, disease progression, and treatment response [1]. Severe uncontrolled asthmatics make up approximately 5-10% of the population of asthmatics, yet they consume around 50% of the treatment resources [2]. Spirometric tests are incapable of exposing the underlying pathologies or phenotypes which combine within an individual to produce the asthmatic disease. In contrast specific biomarkers may enable more accurate sub-phenotyping of disease by indicating the pathology within an individual, and assist clinicians to better tailor the type and/or dose of therapy. In this way biomarkers have the potential to guide more effective personalized treatment regimes in asthma.

© 2012 Dorscheid et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Dorscheid et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1.2. The causes of asthma**

Several studies have used lung biopsies taken from non-asthmatic patients and those who died from fatal asthma to show the airways of asthmatics possess characteristic structural differences, shown in Figure 1. These include but are not limited to; increased eosinophil numbers in asthmatics, and areas of focal bronchial epithelial denudation [3]. The contents of the bronchial lumen can also be examined using the bronchoalveolar lavage (BAL) technique, in which warm saline is instilled into a segmental bronchus of a patient and removed. BAL fluid-based studies confirmed the previous research that inflammatory cell influx and epithelial damage occur in the airways of asthmatics [3] [4]. It is a point of some debate as to whether the epithelial damage is caused by inflammation, is a cause of inflammation, or whether epithelial damage and airway inflammation are concurrent but unrelated processes. We shall discuss the potential role of the epithelium and epithelial repair in asthma in more detail later.

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 189

atopic asthmatics triggered an increase of IL-13 protein and transcript in bronchoalveolar lavage, and the source was identified as mononuclear inflammatory cells [6]. Experiments in small mammals and *in vitro* studies using human cells, have demonstrated IL-13 triggers many of the pathophysiological characteristics of asthma, independent of the IgE and eosinophil inflammatory mechanisms classically associated with allergic disease [7] [8]. Genetic studies in humans have demonstrated the gene loci for IL-13 and the IL-4 receptor portion of the IL-13Rα1 receptor have a strong genetic association to asthma susceptibility and severity [9]. IL-13 can act directly on the airway epithelium in various ways; previous studies have demonstrated IL-13 exposure of cultured human airway epithelial cells triggers an matrix-metalloproteinase-7 (MMP-7)-dependent release of the pro-inflammatory chemokine, soluble Fas-ligand, potentially leading to local inflammation and epithelial damage via removal of the protective Fas-ligand-mediated epithelial immune barrier [10]. Mucous hypersecretion is a common observation in asthmatic airways; acute treatment of differentiated cultures of primary human airway epithelial cells with IL-13 induces a hypersecretory phenotype [11], which develops into an asthma-like mucous hyperplasia with a 5-10-fold increase in goblet cell density and MUC5AC (the predominant airway mucin) detection after 14 day treatment [12]. Murine studies confirm IL-13 is required and sufficient to directly induce epithelial mucous hyperplasia *in vivo* in the absence of intermediate inflammatory cells [13]. Mucins including MUC5AC are highly glycosylated proteins, it thus follows that increased mucous production will necessitate an increase in the activity of associated transferase enzymes. In the H292 airway epithelial cell line, IL-13 treatment has been shown to increase the activity of the M and L isoforms of core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) via activation of a JAK/STAT signaling pathway [14]. Thus, IL-13 acts directly on the airway epithelium to trigger development of an asthmalike phenotype, at least in part by increasing protein glycosylation via upregulation of

Asthma is rarely fatal but often leads to significant patient morbidity. For over thirty years the mainstay therapy for asthma has been inhaled β2-agonists (βA), which relax the airway smooth muscles, causing bronchodilation, and inhaled glucocorticosteroids (i.e. steroids), which ameliorate airway inflammation and reduce the risk of asthma exacerbations. Steroids are anti-inflammatory drugs but also have many unwanted side-effects, particularly at high doses, including; weight-gain due to changes in body metabolism, and a reduction in bone mineral density, potentially leading to brittle bones. Therefore the decision to treat a patient with steroids should not be taken lightly. Inhaled β2-agonists (s) are the most effective bronchodilators available and their main mechanism of action is via the relaxation of airway smooth muscle [15]. βAs interact with G-protein-coupled cell surface β2-adrenoceptors (β2AR) on bronchial smooth muscle cells and activate adenyl cyclase, causing a rise in intracellular cAMP, this results in relaxation of the smooth muscle and bronchodilation. Long term βA treatment can result in a loss of response which shows variation between individuals. Desensitisation due to chronic βA treatment is caused by a

glucosyltransferase activity.

**1.5. Current treatments for asthma**

### **1.3. Inflammation in asthma**

The rationale for treating severe asthmatics with steroids is centred on the assumption that the disease is driven by uncontrolled airway inflammation. The classical paradigm of the acute inflammatory response in allergic asthma is briefly; inhaled allergenic antigens are captured by professional antigen-presenting dendritic cells, macrophages, or epithelial cells. Antigens presented by these cells are recognized by T-cells which proliferate and differentiate. Primed Th2 cells bind to B cells and release cytokines including triggering maturation of antigen-specific B cell populations into plasma cells. Plasma B cells release antigen-specific IgE that binds to IgE-receptors on mast cells in the airways, causing release of histamine-containing granules. Extracellular histamine released from mast cells binds to membrane receptors on airway smooth muscle cells triggering a rise in intracellular calcium, muscle contraction and airway narrowing. Although this paradigm has gained acceptance as an important mechanism in asthma, there is significant variation in specific cell types involved between individuals. In the setting of chronic asthma, eosinophils are thought to play a major role in maintaining airway inflammation in the long-term. Neutrophils are occasionally the predominate inflammatory cell present in the airways of chronic asthmatics, suggesting multiple underlying pathologies of disease. Neutrophilic vs. eosinophilic asthma are clinically indistinguishable by pulmonary function testing, but such pathological variation may have important implications regarding treatment as patients with "neutrophilic asthma" tend to be relatively unresponsive to steroid treatment [5]. A rapid and definitive method for determining the nature of airways inflammation in a diagnosed asthmatic is therefore needed to guide the clinician's selection of therapy.

#### **1.4. IL-13 and asthma**

Interleukin-13 (IL-13) is a pleiotropic Th2 cytokine secreted from a variety of inflammatory cells and structural cells, including the airway epithelium. IL-13 is upregulated in the airways of patients with allergic asthma exposed to allergen; segmental allergen challenge of atopic asthmatics triggered an increase of IL-13 protein and transcript in bronchoalveolar lavage, and the source was identified as mononuclear inflammatory cells [6]. Experiments in small mammals and *in vitro* studies using human cells, have demonstrated IL-13 triggers many of the pathophysiological characteristics of asthma, independent of the IgE and eosinophil inflammatory mechanisms classically associated with allergic disease [7] [8]. Genetic studies in humans have demonstrated the gene loci for IL-13 and the IL-4 receptor portion of the IL-13Rα1 receptor have a strong genetic association to asthma susceptibility and severity [9]. IL-13 can act directly on the airway epithelium in various ways; previous studies have demonstrated IL-13 exposure of cultured human airway epithelial cells triggers an matrix-metalloproteinase-7 (MMP-7)-dependent release of the pro-inflammatory chemokine, soluble Fas-ligand, potentially leading to local inflammation and epithelial damage via removal of the protective Fas-ligand-mediated epithelial immune barrier [10]. Mucous hypersecretion is a common observation in asthmatic airways; acute treatment of differentiated cultures of primary human airway epithelial cells with IL-13 induces a hypersecretory phenotype [11], which develops into an asthma-like mucous hyperplasia with a 5-10-fold increase in goblet cell density and MUC5AC (the predominant airway mucin) detection after 14 day treatment [12]. Murine studies confirm IL-13 is required and sufficient to directly induce epithelial mucous hyperplasia *in vivo* in the absence of intermediate inflammatory cells [13]. Mucins including MUC5AC are highly glycosylated proteins, it thus follows that increased mucous production will necessitate an increase in the activity of associated transferase enzymes. In the H292 airway epithelial cell line, IL-13 treatment has been shown to increase the activity of the M and L isoforms of core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) via activation of a JAK/STAT signaling pathway [14]. Thus, IL-13 acts directly on the airway epithelium to trigger development of an asthmalike phenotype, at least in part by increasing protein glycosylation via upregulation of glucosyltransferase activity.

#### **1.5. Current treatments for asthma**

188 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

Several studies have used lung biopsies taken from non-asthmatic patients and those who died from fatal asthma to show the airways of asthmatics possess characteristic structural differences, shown in Figure 1. These include but are not limited to; increased eosinophil numbers in asthmatics, and areas of focal bronchial epithelial denudation [3]. The contents of the bronchial lumen can also be examined using the bronchoalveolar lavage (BAL) technique, in which warm saline is instilled into a segmental bronchus of a patient and removed. BAL fluid-based studies confirmed the previous research that inflammatory cell influx and epithelial damage occur in the airways of asthmatics [3] [4]. It is a point of some debate as to whether the epithelial damage is caused by inflammation, is a cause of inflammation, or whether epithelial damage and airway inflammation are concurrent but unrelated processes. We shall discuss the potential role of the epithelium and epithelial

The rationale for treating severe asthmatics with steroids is centred on the assumption that the disease is driven by uncontrolled airway inflammation. The classical paradigm of the acute inflammatory response in allergic asthma is briefly; inhaled allergenic antigens are captured by professional antigen-presenting dendritic cells, macrophages, or epithelial cells. Antigens presented by these cells are recognized by T-cells which proliferate and differentiate. Primed Th2 cells bind to B cells and release cytokines including triggering maturation of antigen-specific B cell populations into plasma cells. Plasma B cells release antigen-specific IgE that binds to IgE-receptors on mast cells in the airways, causing release of histamine-containing granules. Extracellular histamine released from mast cells binds to membrane receptors on airway smooth muscle cells triggering a rise in intracellular calcium, muscle contraction and airway narrowing. Although this paradigm has gained acceptance as an important mechanism in asthma, there is significant variation in specific cell types involved between individuals. In the setting of chronic asthma, eosinophils are thought to play a major role in maintaining airway inflammation in the long-term. Neutrophils are occasionally the predominate inflammatory cell present in the airways of chronic asthmatics, suggesting multiple underlying pathologies of disease. Neutrophilic vs. eosinophilic asthma are clinically indistinguishable by pulmonary function testing, but such pathological variation may have important implications regarding treatment as patients with "neutrophilic asthma" tend to be relatively unresponsive to steroid treatment [5]. A rapid and definitive method for determining the nature of airways inflammation in a

diagnosed asthmatic is therefore needed to guide the clinician's selection of therapy.

Interleukin-13 (IL-13) is a pleiotropic Th2 cytokine secreted from a variety of inflammatory cells and structural cells, including the airway epithelium. IL-13 is upregulated in the airways of patients with allergic asthma exposed to allergen; segmental allergen challenge of

**1.2. The causes of asthma**

repair in asthma in more detail later.

**1.3. Inflammation in asthma**

**1.4. IL-13 and asthma**

Asthma is rarely fatal but often leads to significant patient morbidity. For over thirty years the mainstay therapy for asthma has been inhaled β2-agonists (βA), which relax the airway smooth muscles, causing bronchodilation, and inhaled glucocorticosteroids (i.e. steroids), which ameliorate airway inflammation and reduce the risk of asthma exacerbations. Steroids are anti-inflammatory drugs but also have many unwanted side-effects, particularly at high doses, including; weight-gain due to changes in body metabolism, and a reduction in bone mineral density, potentially leading to brittle bones. Therefore the decision to treat a patient with steroids should not be taken lightly. Inhaled β2-agonists (s) are the most effective bronchodilators available and their main mechanism of action is via the relaxation of airway smooth muscle [15]. βAs interact with G-protein-coupled cell surface β2-adrenoceptors (β2AR) on bronchial smooth muscle cells and activate adenyl cyclase, causing a rise in intracellular cAMP, this results in relaxation of the smooth muscle and bronchodilation. Long term βA treatment can result in a loss of response which shows variation between individuals. Desensitisation due to chronic βA treatment is caused by a

down-regulation of receptor transcription, secondary to a reduced activity of the CREB (cyclic AMP response element binding protein) transcription factor [15]. The β2 adrenoceptor gene possesses at least three possible GRE binding sites for the activated GCR, and GCs have been shown to increase β2AR transcription two-fold in human lung preparations *in vitro* and also in rat lungs *in vivo*. The downregulation of β2AR transcription resulting from long-term βA treatment can therefore be reversed by a GC, so there is no net change in the level of β2ARs expressed in the bronchial epithelium or airway smooth muscle of animals treated with a combination of GCs and βAs [15]. The very large β2AR reservoir in airway smooth muscle cells may mean that this upregulation by GCs is functionally unimportant *in vivo* [15]. Many asthmatics who are unresponsive to high doses of steroids show a marked improvement in symptoms when given steroids with βA together. Thus there may be a true synergy between the two classes of drugs as β2-adrenoceptor agonists enhance the function of GCs both *in vitro* and *in vivo*, possibly by increasing the nuclear localisation of activated GCRs. Low-dose theophylline has been prescribed as an asthma treatment for many years, however it has now been relegated to a third-line therapy according to the global asthma guidelines (reviewed in [16]). Theophylline is a weak, nonspecific cyclic nucleotide phosphodiesterase (PDE) inhibitor, and recently these PDE inhibitory characteristics have re-ignited interest in this drug as a possible asthma treatment. IL-13, Asthma and Glycosylation in Airway Epithelial Repair 191

Carbohydrate decoration of proteins has been associated with asthmatic disease. Two casecontrol studies have investigated the role of the histoblood group antigens; H(O), A, B, or AB in asthma susceptibility. They found in human subjects the O-secretor mucin glycan (Hantigen) phenotype is associated with an increased susceptibility to recurrent asthma

From the level of the bronchioles upwards, the stratified epithelium lining the airway lumen rests on a basal lamina or 'true' basement membrane, a specialised 'mat' of extracellular matrix proteins. Below the basal lamina is a layer of collagenous matrix, termed the reticular basement membrane, in which fibroblasts are embedded in a sporadic arrangement. Farther below the fibroblastic layer are arranged bundles of smooth muscle myocytes, blood vessels

Although asthma is considered an inflammatory disease, there are many structural changes in the airways. Figure 1. shows cross-sections through the large airways of two patients, one normal and one severe asthmatic. The Movat's pentachrome stain clearly highlights the various architectural remodeling events occurring in the asthmatic airway. Obstruction of the airways from excessive mucus production is a common finding in severe asthmatics, blue staining in the epithelium and the lumen demonstrates mucous cell hyperplasia, with excessive mucus deposition into the airway. Under the asthmatic epithelium a thicker basement membrane is present which contains several different extracellular matrix (ECM) factors (including tenascin-C) compared to normals [22]. Deeper into the airway, red-stained muscle mass is increased in the asthmatic owing to a combination of smooth muscle hypertrophy and hyperplasia (reviewed in [23]). The airways of severe asthmatics also demonstrate a fibrotic response with increased connective tissue deposition and fibroblast and myofibroblast proliferation. Opinion is divided as to whether inflammation precedes airway remodeling, or whether the two occur in parallel (reviewed in [15, 24, 25]). Evidence tends to favour the latter because; firstly, remodeling occurs very early on in the disease, and in some cases in the absence of inflammation [26], secondly, there is only a weak link between airway inflammation and symptoms [27], and thirdly, epidemiological data demonstrate steroids do not work in all asthmatics [5]. In reality it is likely effective

**1.6. Carbohydrates and asthma**

**2. Normal airway structure**

and afferent nerve endings.

**2.1. Structural changes to the airway in asthma**

therapies will need to target both airway inflammation and remodeling.

The bronchial epithelium lines the inner wall of the respiratory tract in a continuous layer; it forms the interface between inspired air and the internal milieu as well as being the primary target for inhaled respiratory drugs. In the larger airways and down the respiratory tree to

**2.2. Airway epithelial structure and function**

exacerbations [21].

Monoclonal antibodies have gained recent credence as a potential means to target therapy against specific receptors and signalling pathways. Omalizumab, a biologic agent that binds to IgE, has shown some promise as a means of controlling some difficult to treat asthma patients who are poorly controlled with standard inhaled steroid and β-agonist combination therapy (reviewed in [17]). Interleukin-5 (IL-5) is an important cytokine for eosinophil differentiation, maturation, migration into the circulation and survival. Various studies have implicated the eosinophil as being the primary cell responsible for airway hyperresponsiveness in asthma, thus anti-IL-5 antibodies have been developed (such as mepolizumab) with the intention of reducing airways hyperresponsiveness by preventing eosinophil recruitment and survival in the airway. Clinical trials have demonstrated such inhaled anti-IL-5 therapy does indeed reduce airway eosinophilia, but does not have a clinically-beneficial effect on lung function [18, 19]. Considering the highly important role IL-13 is thought to play in asthma, it is unsurprising that therapies are being developed to specifically target IL-13. Lebrikizumab, an anti-IL-13 monoclonal antibody, has just completed phase II clinical trials in which patients with poorly-controlled asthma were given the drug subcutaneously at 4 weekly intervals for 6 months [20]. This study found anti-IL-13 therapy did significantly improve lung function, but only in a sub-group of asthma patients who exhibited high serum periostin levels, a surrogate marker of high airway IL-13 [20]. These studies demonstrate highly-targeted therapies may be useful in asthma, but each individual drug will probably only prove effective in a specific sub-group of asthmatic patients. The potential for such personalised medicine highlights the importance for accurate phenotyping of asthmatic individuals, discussed in the biomarkers section of this chapter (Section 5).

### **1.6. Carbohydrates and asthma**

190 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

section of this chapter (Section 5).

down-regulation of receptor transcription, secondary to a reduced activity of the CREB (cyclic AMP response element binding protein) transcription factor [15]. The β2 adrenoceptor gene possesses at least three possible GRE binding sites for the activated GCR, and GCs have been shown to increase β2AR transcription two-fold in human lung preparations *in vitro* and also in rat lungs *in vivo*. The downregulation of β2AR transcription resulting from long-term βA treatment can therefore be reversed by a GC, so there is no net change in the level of β2ARs expressed in the bronchial epithelium or airway smooth muscle of animals treated with a combination of GCs and βAs [15]. The very large β2AR reservoir in airway smooth muscle cells may mean that this upregulation by GCs is functionally unimportant *in vivo* [15]. Many asthmatics who are unresponsive to high doses of steroids show a marked improvement in symptoms when given steroids with βA together. Thus there may be a true synergy between the two classes of drugs as β2-adrenoceptor agonists enhance the function of GCs both *in vitro* and *in vivo*, possibly by increasing the nuclear localisation of activated GCRs. Low-dose theophylline has been prescribed as an asthma treatment for many years, however it has now been relegated to a third-line therapy according to the global asthma guidelines (reviewed in [16]). Theophylline is a weak, nonspecific cyclic nucleotide phosphodiesterase (PDE) inhibitor, and recently these PDE inhibitory characteristics have re-ignited interest in this drug as a possible asthma treatment. Monoclonal antibodies have gained recent credence as a potential means to target therapy against specific receptors and signalling pathways. Omalizumab, a biologic agent that binds to IgE, has shown some promise as a means of controlling some difficult to treat asthma patients who are poorly controlled with standard inhaled steroid and β-agonist combination therapy (reviewed in [17]). Interleukin-5 (IL-5) is an important cytokine for eosinophil differentiation, maturation, migration into the circulation and survival. Various studies have implicated the eosinophil as being the primary cell responsible for airway hyperresponsiveness in asthma, thus anti-IL-5 antibodies have been developed (such as mepolizumab) with the intention of reducing airways hyperresponsiveness by preventing eosinophil recruitment and survival in the airway. Clinical trials have demonstrated such inhaled anti-IL-5 therapy does indeed reduce airway eosinophilia, but does not have a clinically-beneficial effect on lung function [18, 19]. Considering the highly important role IL-13 is thought to play in asthma, it is unsurprising that therapies are being developed to specifically target IL-13. Lebrikizumab, an anti-IL-13 monoclonal antibody, has just completed phase II clinical trials in which patients with poorly-controlled asthma were given the drug subcutaneously at 4 weekly intervals for 6 months [20]. This study found anti-IL-13 therapy did significantly improve lung function, but only in a sub-group of asthma patients who exhibited high serum periostin levels, a surrogate marker of high airway IL-13 [20]. These studies demonstrate highly-targeted therapies may be useful in asthma, but each individual drug will probably only prove effective in a specific sub-group of asthmatic patients. The potential for such personalised medicine highlights the importance for accurate phenotyping of asthmatic individuals, discussed in the biomarkers

Carbohydrate decoration of proteins has been associated with asthmatic disease. Two casecontrol studies have investigated the role of the histoblood group antigens; H(O), A, B, or AB in asthma susceptibility. They found in human subjects the O-secretor mucin glycan (Hantigen) phenotype is associated with an increased susceptibility to recurrent asthma exacerbations [21].

## **2. Normal airway structure**

From the level of the bronchioles upwards, the stratified epithelium lining the airway lumen rests on a basal lamina or 'true' basement membrane, a specialised 'mat' of extracellular matrix proteins. Below the basal lamina is a layer of collagenous matrix, termed the reticular basement membrane, in which fibroblasts are embedded in a sporadic arrangement. Farther below the fibroblastic layer are arranged bundles of smooth muscle myocytes, blood vessels and afferent nerve endings.

## **2.1. Structural changes to the airway in asthma**

Although asthma is considered an inflammatory disease, there are many structural changes in the airways. Figure 1. shows cross-sections through the large airways of two patients, one normal and one severe asthmatic. The Movat's pentachrome stain clearly highlights the various architectural remodeling events occurring in the asthmatic airway. Obstruction of the airways from excessive mucus production is a common finding in severe asthmatics, blue staining in the epithelium and the lumen demonstrates mucous cell hyperplasia, with excessive mucus deposition into the airway. Under the asthmatic epithelium a thicker basement membrane is present which contains several different extracellular matrix (ECM) factors (including tenascin-C) compared to normals [22]. Deeper into the airway, red-stained muscle mass is increased in the asthmatic owing to a combination of smooth muscle hypertrophy and hyperplasia (reviewed in [23]). The airways of severe asthmatics also demonstrate a fibrotic response with increased connective tissue deposition and fibroblast and myofibroblast proliferation. Opinion is divided as to whether inflammation precedes airway remodeling, or whether the two occur in parallel (reviewed in [15, 24, 25]). Evidence tends to favour the latter because; firstly, remodeling occurs very early on in the disease, and in some cases in the absence of inflammation [26], secondly, there is only a weak link between airway inflammation and symptoms [27], and thirdly, epidemiological data demonstrate steroids do not work in all asthmatics [5]. In reality it is likely effective therapies will need to target both airway inflammation and remodeling.

## **2.2. Airway epithelial structure and function**

The bronchial epithelium lines the inner wall of the respiratory tract in a continuous layer; it forms the interface between inspired air and the internal milieu as well as being the primary target for inhaled respiratory drugs. In the larger airways and down the respiratory tree to

the level of the bronchioles the epithelium is at least two cells thick (reviewed. in [28]). Although there are at least eight morphologically and functionally distinct epithelial cell types present in the respiratory tract, they can be classified into three main groups; basal, ciliated columnar, and secretory columnar (reviewed in [29]).

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 193

[4, 30]. Historically the basal cell has been considered to be the stem cell of the bronchial epithelium (reviewed in [31]), however animal studies suggest there may be alternative stem cells located in the airway epithelium, including secretory cells, and cells located at specialised niches including the broncho-alveolar junction and the neuroepithelial bodies.

Columnar epithelial cells lie above the basal cell layer and line the airway lumen, in direct contact with the inspired air. Ciliated cells are terminally differentiated columnar cells and are the most common cell-type in the bronchial epithelium, accounting for around 50% of all epithelial cells and 80% of terminally-differentiated apical cells in the human trachea [32], reviewed in [29]. Their main function is the removal of particulate matter by means of the mucociliary pathway. Ciliated cells arise from either a basal or secretory cell pre-cursor and possess around 300 cilia per cell, directly beneath the cilia are observed numerous mitochondria and a dense microtubule system, reflecting the high metabolic demands of

Secretory cells are also present at the apical surface, comprising 15 to 20% of the normal tracheobronchial epithelium [32]. In the larger airways the mucus-secreting goblet cell represents the predominant secretory cell and is the main source of airway mucus. These cells are characterised by membrane-bound electron-lucent acidic-mucin granules which are released into the airway lumen to trap inhaled particles and pathogens, prior to removal from the respiratory tract by coordinated cilia beating. Serous cells and Clara cells are relatively rare secretory cells in the larger airways present in large airways, but Clara cells are the main secretory epithelial cell type of the bronchioles, particularly the most distal bronchioles (reviewed in [34]). Several studies in rodents indicate the progenitor cell of the bronchial epithelium is not the basal cell but it is in fact the non-ciliated, secretory columnar cell. Both goblet and clara cells have been observed to undergo de-differentiation and

The airway epithelial surface is covered by a layer of airway surface liquid (ASL), mainly of epithelial origin. A layer of mucins form a mucous layer that overlies the thin watery periciliary layer (PCL). The predominant mucins in the airways are MUC5AC and MUC5B secreted by goblet cells and submucosal glands respectively. Mucins are highly glycosylated proteins; 70-80% of their molecular weight are carbohydrate, and the structural characteristics of the mucous layer depends on interaction between the carbohydrate sidechains of mucin proteins. Studies of airway mucins in disease, demonstrate mucins are both oversulphated and hyper-sialylated in patients with cystic fibrosis and in chronic bronchitis patients with significant infections [39]. Pseudomonas aeruginosa, the pathogen responsible for the majority of morbidity and mortality in cystic fibrosis patients, uses the sialylated and sulphated Lewis-x determinants as attachment sites. Thus mucin glycosylation patterns may

**Columnar ciliated cells**

particle clearance [29] [33].

proliferation during *in vivo* wound repair [4] [35-38].

**2.3. Airway epithelial carbohydrate expression**

**Secretory cells**

**Figure 1. The airways in asthma undergo significant structural remodeling.** Medium-sized airways from a normal and severe asthmatic patient were sectioned and stained using Movat's pentachrome stain. The epithelium (Ep) in asthma shows mucous hyperplasia and hyper secretion (blue), and significant basement membrane (Bm) thickening. Smooth muscle (Sm) volume is also increased in asthma. Bv = blood vessel. Scale bar = 100µm.

#### **Basal cells**

Basal epithelial cells are pyramidal-shaped cells with a low cytoplasm to nucleus ratio, although found throughout the airway, their contribution to epithelial volume decreases with airway size (reviewed in [29]). Basal cells are anchored to the underlying basement membrane and to other cells via specialised adhesion structures discussed below (Figure 2.) [4, 30]. Historically the basal cell has been considered to be the stem cell of the bronchial epithelium (reviewed in [31]), however animal studies suggest there may be alternative stem cells located in the airway epithelium, including secretory cells, and cells located at specialised niches including the broncho-alveolar junction and the neuroepithelial bodies.

#### **Columnar ciliated cells**

192 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

ciliated columnar, and secretory columnar (reviewed in [29]).

the level of the bronchioles the epithelium is at least two cells thick (reviewed. in [28]). Although there are at least eight morphologically and functionally distinct epithelial cell types present in the respiratory tract, they can be classified into three main groups; basal,

**Figure 1. The airways in asthma undergo significant structural remodeling.** Medium-sized airways from a normal and severe asthmatic patient were sectioned and stained using Movat's pentachrome stain. The epithelium (Ep) in asthma shows mucous hyperplasia and hyper secretion (blue), and significant basement membrane (Bm) thickening. Smooth muscle (Sm) volume is also increased in

Basal epithelial cells are pyramidal-shaped cells with a low cytoplasm to nucleus ratio, although found throughout the airway, their contribution to epithelial volume decreases with airway size (reviewed in [29]). Basal cells are anchored to the underlying basement membrane and to other cells via specialised adhesion structures discussed below (Figure 2.)

asthma. Bv = blood vessel. Scale bar = 100µm.

**Basal cells**

Columnar epithelial cells lie above the basal cell layer and line the airway lumen, in direct contact with the inspired air. Ciliated cells are terminally differentiated columnar cells and are the most common cell-type in the bronchial epithelium, accounting for around 50% of all epithelial cells and 80% of terminally-differentiated apical cells in the human trachea [32], reviewed in [29]. Their main function is the removal of particulate matter by means of the mucociliary pathway. Ciliated cells arise from either a basal or secretory cell pre-cursor and possess around 300 cilia per cell, directly beneath the cilia are observed numerous mitochondria and a dense microtubule system, reflecting the high metabolic demands of particle clearance [29] [33].

#### **Secretory cells**

Secretory cells are also present at the apical surface, comprising 15 to 20% of the normal tracheobronchial epithelium [32]. In the larger airways the mucus-secreting goblet cell represents the predominant secretory cell and is the main source of airway mucus. These cells are characterised by membrane-bound electron-lucent acidic-mucin granules which are released into the airway lumen to trap inhaled particles and pathogens, prior to removal from the respiratory tract by coordinated cilia beating. Serous cells and Clara cells are relatively rare secretory cells in the larger airways present in large airways, but Clara cells are the main secretory epithelial cell type of the bronchioles, particularly the most distal bronchioles (reviewed in [34]). Several studies in rodents indicate the progenitor cell of the bronchial epithelium is not the basal cell but it is in fact the non-ciliated, secretory columnar cell. Both goblet and clara cells have been observed to undergo de-differentiation and proliferation during *in vivo* wound repair [4] [35-38].

#### **2.3. Airway epithelial carbohydrate expression**

The airway epithelial surface is covered by a layer of airway surface liquid (ASL), mainly of epithelial origin. A layer of mucins form a mucous layer that overlies the thin watery periciliary layer (PCL). The predominant mucins in the airways are MUC5AC and MUC5B secreted by goblet cells and submucosal glands respectively. Mucins are highly glycosylated proteins; 70-80% of their molecular weight are carbohydrate, and the structural characteristics of the mucous layer depends on interaction between the carbohydrate sidechains of mucin proteins. Studies of airway mucins in disease, demonstrate mucins are both oversulphated and hyper-sialylated in patients with cystic fibrosis and in chronic bronchitis patients with significant infections [39]. Pseudomonas aeruginosa, the pathogen responsible for the majority of morbidity and mortality in cystic fibrosis patients, uses the sialylated and sulphated Lewis-x determinants as attachment sites. Thus mucin glycosylation patterns may significantly affect infection in the airway. Specific lectin-binding assays have been used to characterise carbohydrate expression patterns in differentiated human airway epithelium [40]. Out of 38 lectin probes tested, seven bound specifically to basal cells, seven to columnar cells, and three specifically labelled secretory cells. The 1HAEo- and 16HBE14o- airway epithelial cell lines were also probed with the same lectin panel, revealing identical carbohydrate expression to the basal epithelial cells in the human tissue [40]. Thus the different cell types in the pseudostratified epithelium of the airways, express specific patterns of carbohydrates, possibly reflecting their different functions.

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 195

airway lumen. Dimeric secretory IgA (sIgA) is released into the airway lumen as a 1:1 combination of dIgA and a portion of the pIgR known as secretory component (SC), which protects the IgA from digestion by proteases on the mucosal surface [40, 41]. SC is a highly glycosylated protein containing 15% N-linked carbohydrate. Glycosylated SC binds IL-8, inhibiting IL-8-mediated neutrophil chemotaxis and transendothelial migration. This interaction is dependent on SC glycosylation state as de-glycosylation with peptide N-

**Figure 2. Adhesion mechanisms in the human bronchial epithelium.** Tight junctions seal the lateral apices of columnar cells which adhere to each other via adherens junctions. Desmosomes mediate basalbasal and columnar-basal cell attachments, whilst hemidesmosomes anchor basal cells to the basal

All epithelial tissues are regularly damaged as a consequence of exposure to environmental insults. Rapid repair following injury is crucial for restoring adequate barrier function, with subsequent cellular differentiation required to regenerate normal epithelial structure and function. The repair processes in all epithelial types have common elements producing an

glycosidase F abolishes the SC-IL-8 complex [42].

lamina. (Adapted from Roche et al. [30]).

**3. Epithelial injury and repair**

## **2.4. Airway epithelial functions**

The primary function of the bronchial epithelium is to serve as a continuous physical barrier and as such it functions as part of the non-specific immune system, preventing pollutants, bacteria, viruses, allergens, and other potentially noxious substances transferring from inspired air into the underlying mesenchyme. This defence is mediated by several adhesive mechanisms which have been elucidated through transmission electron microscopy and immunohistochemical studies of bronchial epithelial biopsies [4] [30] (Figure 2.). Belt-like tight junctions (zonula occludens) seal the lateral apices of columnar cells, regulating paracellular transport, whilst columnar cells adhere to each other via classical E-cadherin mediated adherens junctions [30]. Desmosomal cadherins mediate strong cell-cell adhesion and desmosomes are present between basal cells and particularly at the junction of basal and columnar cell layers [30]. Basal cells are in turn anchored to the basement membrane via specialised integrin-mediated cell-ECM junctions. Integrin heterodimers function by dynamically linking the contractile machinery of the cell's cytoskeletal network to the external matrix at specialised sites termed focal adhesions or focal contacts. The airway epithelium expresses several integrin heterodimers, including the α6β4 integrin which binds to laminin-5 in the basement membrane forming a hemidesmosome anchor with the ECM.

The bronchial epithelium is more than a passive barrier; it performs a variety of roles allowing it to function as a dynamic regulator of the innate immune system [39]. The mucociliary pathway represents the principle mechanism of particle clearance in the airway. Inhaled particles and pathogens (bacteria, viruses) are trapped in mucus prior to removal from the respiratory tract by coordinated cilia beating. In addition to producing mucus, secretory cells are the source of a variety of mediators capable of having direct effects on inhaled noxious agents; including anti-oxidants and a variety of anti-bacterial agents; lactoferrin, lysozyme, β-defensins and also opsonins, components of the complement system that coat bacteria to facilitate phagocytosis by macrophages (reviewed in [39]). Various immunoglobin isotypes are secreted onto mucosal surfaces where they act in a protective manner by opsonising bacteria and other pathogens, rendering them relatively harmless and activating phagocytosis and digestion by tissue macrophages. Allergen-specific IgA is secreted by activated B lymphocytes (plasma cells) in the sub-mucosa, dimeric IgA (dIgA) selectively binds to the polymeric Ig receptor (pIgR) expressed on the basal surface of basal airway epithelial cells. The pIgR-IgA complex is subsequently internalised and is transported to the apical surface via the endosomal pathway ready for secretion into the airway lumen. Dimeric secretory IgA (sIgA) is released into the airway lumen as a 1:1 combination of dIgA and a portion of the pIgR known as secretory component (SC), which protects the IgA from digestion by proteases on the mucosal surface [40, 41]. SC is a highly glycosylated protein containing 15% N-linked carbohydrate. Glycosylated SC binds IL-8, inhibiting IL-8-mediated neutrophil chemotaxis and transendothelial migration. This interaction is dependent on SC glycosylation state as de-glycosylation with peptide Nglycosidase F abolishes the SC-IL-8 complex [42].

**Figure 2. Adhesion mechanisms in the human bronchial epithelium.** Tight junctions seal the lateral apices of columnar cells which adhere to each other via adherens junctions. Desmosomes mediate basalbasal and columnar-basal cell attachments, whilst hemidesmosomes anchor basal cells to the basal lamina. (Adapted from Roche et al. [30]).

## **3. Epithelial injury and repair**

194 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

patterns of carbohydrates, possibly reflecting their different functions.

**2.4. Airway epithelial functions**

significantly affect infection in the airway. Specific lectin-binding assays have been used to characterise carbohydrate expression patterns in differentiated human airway epithelium [40]. Out of 38 lectin probes tested, seven bound specifically to basal cells, seven to columnar cells, and three specifically labelled secretory cells. The 1HAEo- and 16HBE14o- airway epithelial cell lines were also probed with the same lectin panel, revealing identical carbohydrate expression to the basal epithelial cells in the human tissue [40]. Thus the different cell types in the pseudostratified epithelium of the airways, express specific

The primary function of the bronchial epithelium is to serve as a continuous physical barrier and as such it functions as part of the non-specific immune system, preventing pollutants, bacteria, viruses, allergens, and other potentially noxious substances transferring from inspired air into the underlying mesenchyme. This defence is mediated by several adhesive mechanisms which have been elucidated through transmission electron microscopy and immunohistochemical studies of bronchial epithelial biopsies [4] [30] (Figure 2.). Belt-like tight junctions (zonula occludens) seal the lateral apices of columnar cells, regulating paracellular transport, whilst columnar cells adhere to each other via classical E-cadherin mediated adherens junctions [30]. Desmosomal cadherins mediate strong cell-cell adhesion and desmosomes are present between basal cells and particularly at the junction of basal and columnar cell layers [30]. Basal cells are in turn anchored to the basement membrane via specialised integrin-mediated cell-ECM junctions. Integrin heterodimers function by dynamically linking the contractile machinery of the cell's cytoskeletal network to the external matrix at specialised sites termed focal adhesions or focal contacts. The airway epithelium expresses several integrin heterodimers, including the α6β4 integrin which binds to laminin-5 in the basement membrane forming a hemidesmosome anchor with the ECM.

The bronchial epithelium is more than a passive barrier; it performs a variety of roles allowing it to function as a dynamic regulator of the innate immune system [39]. The mucociliary pathway represents the principle mechanism of particle clearance in the airway. Inhaled particles and pathogens (bacteria, viruses) are trapped in mucus prior to removal from the respiratory tract by coordinated cilia beating. In addition to producing mucus, secretory cells are the source of a variety of mediators capable of having direct effects on inhaled noxious agents; including anti-oxidants and a variety of anti-bacterial agents; lactoferrin, lysozyme, β-defensins and also opsonins, components of the complement system that coat bacteria to facilitate phagocytosis by macrophages (reviewed in [39]). Various immunoglobin isotypes are secreted onto mucosal surfaces where they act in a protective manner by opsonising bacteria and other pathogens, rendering them relatively harmless and activating phagocytosis and digestion by tissue macrophages. Allergen-specific IgA is secreted by activated B lymphocytes (plasma cells) in the sub-mucosa, dimeric IgA (dIgA) selectively binds to the polymeric Ig receptor (pIgR) expressed on the basal surface of basal airway epithelial cells. The pIgR-IgA complex is subsequently internalised and is transported to the apical surface via the endosomal pathway ready for secretion into the

All epithelial tissues are regularly damaged as a consequence of exposure to environmental insults. Rapid repair following injury is crucial for restoring adequate barrier function, with subsequent cellular differentiation required to regenerate normal epithelial structure and function. The repair processes in all epithelial types have common elements producing an

orderly progression of events including; cell spreading at the margins of the wound, cell migration into the wound, cell proliferation and finally re-differentiation (Figure 3). *In vivo* investigations of wound repair have demonstrated that migration and proliferation are both critical for the rapid restitution of the bronchial epithelium after injury [35-38]. Hamster tracheal epithelia were mechanically denuded to leave behind a bare (and sometimes damaged) basal lamina and wound repair was monitored until a morphologically normal epithelium was restored. Following epithelial removal, plasma promptly exudes into the injured site from the underlying vasculature to cover the denuded basement membrane, facilitating the binding of serum proteins to cellular receptors that stimulate repair of the damaged epithelium. At 12h post-wounding, viable secretory and basal cells at the wound margins de-differentiate, flatten into a squamous morphology, and migrate at about 0.5µm/min to cover the wound site.

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 197

structure is restored by 120h but some persistent metaplasia persists in larger wounds through 168h post-wounding [38]. Although both secretory and basal cells are involved in epithelial repair, secretory cells play a dominant role in these experiments, consistently demonstrating higher mitosis rates than any other epithelial cell type [35-38]. *In vitro* studies using muco-ciliated human bronchial epithelial cells grown in air-liquid interface (ALI) culture, demonstrate that scrape wound repair is achieved by rapid spreading and migration of cells at the wound edge, followed by proliferation of basal cells outside of the wound area [43]. Acute wound repair speeds were inhibited slightly by the steroid dexamethasone, but the same drug potentiated the ability of long-term ALI cultures to

*In vivo* studies demonstrate epithelial repair is accompanied by a corresponding proliferation of the underlying mesenchyme. Hamster tracheal epithelial damage is followed at 24h by capillary endothelial cell division, and at 36h many fibroblasts were observed to be in mitosis [35-37]. Mesenchymal proliferation persisted for a longer period than the epithelial response and supports several *in vitro* studies demonstrating epithelial damage can influence the phenotype and proliferation of underlying fibroblasts [44, 45].

The patho-physiological changes observed in the asthmatic airway may be due to reactivation of the epithelial-mesenchymal trophic unit (EMTU), triggered by an abnormal epithelium that is held in a repair phenotype [24]. *In vitro* studies support the hypothesis that epithelial-derived factors are capable of altering the phenotype of adjacent mesenchymal cells [43, 44]. The mechanisms involved in initiating and maintaining the epithelial repair response in asthma are largely unknown, however recent studies have demonstrated the airway epithelium in asthmatics is intrinsically altered compared to normals, and that these differences persist for extended periods in culture [45, 46]. There is considerable evidence to suggest the airway epithelium is capable of orchestrating the inflammatory immune response, as following injury, epithelial cells release a variety of proinflammatory cytokines, chemokines and growth factors. Acute inflammation is a necessary aspect of wound repair, however if epithelial damage persists, or the epithelial repair response is not shut-off, then potentially deleterious chronic inflammation may result

Many disorders characterized by impaired re-epithelialization, are not a result of inadequate proliferation, but are due to impaired cell migration over the denuded site [47-49]. Cell migration requires a coordinated, highly complex series of events including; changes to the cell cytoskeleton, modification of the surrounding ECM and modulation of adhesions to ECM substrate and to other cells. The majority of these processes are regulated by the Rholike family of small GTPases including, RhoA, B, C, E, Rac1, 2, 3 and Cdc42 (Figure 3.) (reviewed in [50, 51]) that function by acting as "molecular switches", cycling between an

**3.1. Airway epithelial wound repair is compromised in asthmatics**

repair repeated wounds [43].

(Figure 4.).

**3.2. Mechanisms of cellular migration**

**Figure 3.** Airway epithelial repair involves a series of events including, cell spreading, migration, proliferation and differentiation. Following epithelial injury, neighboring cells flatten, spread and migrate to cover the site of injury. Subsequent proliferation and differentiation restores the epithelial structure and barrier function. The different stages of repair are mediated and facilitated by a diverse array of growth factors, cytokines and cell surface receptors, adhesion molecules, and intracellular enzymes, many of which are glycosylated. These glycans can modulate the function of proteins and lipids thereby regulating the repair process.

Proliferation is also an intrinsic component of epithelial repair. In the aforementioned hamster tracheal injury studies, mitosis rates at 12h post-injury are low (0.4%) but by 24h a wave of proliferation occurs primarily in secretory cells, resulting in a multi-layered epidermoid metaplasia [35]. At 48h post-wounding proliferation rates decrease (although mitosis is still significantly above basal levels), from this time point onwards the upper-most cells of the metaplastic epidermis start to slough off, resulting in a gradual loss of the metaplastic phenotype, with subsequent regeneration of a functional mucociliated phenotype achieved by apical cell re-differentiation. In small wounds normal mucociliary structure is restored by 120h but some persistent metaplasia persists in larger wounds through 168h post-wounding [38]. Although both secretory and basal cells are involved in epithelial repair, secretory cells play a dominant role in these experiments, consistently demonstrating higher mitosis rates than any other epithelial cell type [35-38]. *In vitro* studies using muco-ciliated human bronchial epithelial cells grown in air-liquid interface (ALI) culture, demonstrate that scrape wound repair is achieved by rapid spreading and migration of cells at the wound edge, followed by proliferation of basal cells outside of the wound area [43]. Acute wound repair speeds were inhibited slightly by the steroid dexamethasone, but the same drug potentiated the ability of long-term ALI cultures to repair repeated wounds [43].

*In vivo* studies demonstrate epithelial repair is accompanied by a corresponding proliferation of the underlying mesenchyme. Hamster tracheal epithelial damage is followed at 24h by capillary endothelial cell division, and at 36h many fibroblasts were observed to be in mitosis [35-37]. Mesenchymal proliferation persisted for a longer period than the epithelial response and supports several *in vitro* studies demonstrating epithelial damage can influence the phenotype and proliferation of underlying fibroblasts [44, 45].

## **3.1. Airway epithelial wound repair is compromised in asthmatics**

The patho-physiological changes observed in the asthmatic airway may be due to reactivation of the epithelial-mesenchymal trophic unit (EMTU), triggered by an abnormal epithelium that is held in a repair phenotype [24]. *In vitro* studies support the hypothesis that epithelial-derived factors are capable of altering the phenotype of adjacent mesenchymal cells [43, 44]. The mechanisms involved in initiating and maintaining the epithelial repair response in asthma are largely unknown, however recent studies have demonstrated the airway epithelium in asthmatics is intrinsically altered compared to normals, and that these differences persist for extended periods in culture [45, 46]. There is considerable evidence to suggest the airway epithelium is capable of orchestrating the inflammatory immune response, as following injury, epithelial cells release a variety of proinflammatory cytokines, chemokines and growth factors. Acute inflammation is a necessary aspect of wound repair, however if epithelial damage persists, or the epithelial repair response is not shut-off, then potentially deleterious chronic inflammation may result (Figure 4.).

## **3.2. Mechanisms of cellular migration**

196 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

0.5µm/min to cover the wound site.

lipids thereby regulating the repair process.

orderly progression of events including; cell spreading at the margins of the wound, cell migration into the wound, cell proliferation and finally re-differentiation (Figure 3). *In vivo* investigations of wound repair have demonstrated that migration and proliferation are both critical for the rapid restitution of the bronchial epithelium after injury [35-38]. Hamster tracheal epithelia were mechanically denuded to leave behind a bare (and sometimes damaged) basal lamina and wound repair was monitored until a morphologically normal epithelium was restored. Following epithelial removal, plasma promptly exudes into the injured site from the underlying vasculature to cover the denuded basement membrane, facilitating the binding of serum proteins to cellular receptors that stimulate repair of the damaged epithelium. At 12h post-wounding, viable secretory and basal cells at the wound margins de-differentiate, flatten into a squamous morphology, and migrate at about

**Figure 3.** Airway epithelial repair involves a series of events including, cell spreading, migration, proliferation and differentiation. Following epithelial injury, neighboring cells flatten, spread and migrate to cover the site of injury. Subsequent proliferation and differentiation restores the epithelial structure and barrier function. The different stages of repair are mediated and facilitated by a diverse array of growth factors, cytokines and cell surface receptors, adhesion molecules, and intracellular enzymes, many of which are glycosylated. These glycans can modulate the function of proteins and

Proliferation is also an intrinsic component of epithelial repair. In the aforementioned hamster tracheal injury studies, mitosis rates at 12h post-injury are low (0.4%) but by 24h a wave of proliferation occurs primarily in secretory cells, resulting in a multi-layered epidermoid metaplasia [35]. At 48h post-wounding proliferation rates decrease (although mitosis is still significantly above basal levels), from this time point onwards the upper-most cells of the metaplastic epidermis start to slough off, resulting in a gradual loss of the metaplastic phenotype, with subsequent regeneration of a functional mucociliated phenotype achieved by apical cell re-differentiation. In small wounds normal mucociliary

Many disorders characterized by impaired re-epithelialization, are not a result of inadequate proliferation, but are due to impaired cell migration over the denuded site [47-49]. Cell migration requires a coordinated, highly complex series of events including; changes to the cell cytoskeleton, modification of the surrounding ECM and modulation of adhesions to ECM substrate and to other cells. The majority of these processes are regulated by the Rholike family of small GTPases including, RhoA, B, C, E, Rac1, 2, 3 and Cdc42 (Figure 3.) (reviewed in [50, 51]) that function by acting as "molecular switches", cycling between an

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 199

**Figure 5. Cellular processes occurring during migration.** The transition from a stationary state to a migratory phenotype involves several distinct mechanisms, Rho-like GTPases regulate the majority of these processes and the individual GTPases most directly linked to each are: (1.) Rac-dependent actin (blue) polymerisation leads to lamellipodium extension at the leading edge of the cell, (2.) new ECM adhesions are formed at the lamellipodium via focal contacts (red), also regulated by Rac, (3.) cell body contraction is dependent on actomyosin contraction (blue arrows) and is dependent on Rho activity, (4.)

tail detachment is possibly affected by GTPase activity but the protease calpain is important for degradation of focal adhesions at the rear of migrating cells, (5.) cadherin-mediated cell-cell adhesions (green) are dependent on Rho and Rac activity and are often downregulated during migration. MMPmediated ECM degradation is necessary for migration, even in cells migrating over a basement

**3.3. The effect of intracellular protein glycosylation on epithelial repair**

The activity of the Rho-GTPase family is highly regulated by glycosylation. *Clostridium difficile* toxins A and B (TcdA and TcdB) are major virulence factors for the induction of antibiotic-associated diarrhea and pseudomembranous colitis. These toxins possess potent

Several *in vitro* studies have demonstrated activity of the RhoGTPase family is essential for airway epithelial wound repair; activation of protein kinase A (PKA) facilitates scrape wound closure in cultured bovine airway epithelial cells via inhibition of Rho activity and decreased focal contact attachments [57]. The 16HBE human airway epithelial cell line has been used to demonstrate coordination of RhoA and Rac1 activities are vital for efficient wound closure [58]. Detachment of the trailing edge is often a limiting factor of cellular migration speed, detachment is possibly affected by GTPase activity but the protease calpain is important for degradation of focal adhesions at the rear of migrating cells. Rho-GTPases are also involved in cell proliferation during airway epithelial repair; the scaffold IQ domain GTPase-activating protein (IQGAP1), an effector of Rho GTPases, is upregulated during 16HBE scrape wound repair and facilitates closure by increasing via a ß-cateninmediated transcription of genes involved in cell cycle regulation such as CyclinD1 [59, 60].

membrane.

**Figure 4. Injury and repair cycle of the airway epithelium in asthma**. Complete repair requires glycosylation as a means of regulation of essential elements. Aberrant glycosylation would result in a defect in the mechanisms of repair, the accumulation of epithelial damage and persistent airway inflammation. Modified from Davies [46].

inactive GDP-bound state and an active GTP-bound form at the plasma membrane [52]. GTPases are able to regulate the transmission of signals from cell surface receptors, such as integrin-mediated cell-ECM adhesion, or growth factor ligation, to downstream intracellular signalling pathways, influencing the cell cytoskeleton, gene transcription, and the cell cycle (reviewed in [53]). Rho induces formation of stress fibres, integrin-mediated focal adhesions, and actomyosin-mediated cell body contraction [54], Rac-dependent actin polymerisation leads to lamellipodium extension and membrane ruffles at the leading edge of the cell [55], and Cdc42 induces formation of microspikes/filopodia [56].

**Figure 4. Injury and repair cycle of the airway epithelium in asthma**. Complete repair requires glycosylation as a means of regulation of essential elements. Aberrant glycosylation would result in a defect in the mechanisms of repair, the accumulation of epithelial damage and persistent airway

inactive GDP-bound state and an active GTP-bound form at the plasma membrane [52]. GTPases are able to regulate the transmission of signals from cell surface receptors, such as integrin-mediated cell-ECM adhesion, or growth factor ligation, to downstream intracellular signalling pathways, influencing the cell cytoskeleton, gene transcription, and the cell cycle (reviewed in [53]). Rho induces formation of stress fibres, integrin-mediated focal adhesions, and actomyosin-mediated cell body contraction [54], Rac-dependent actin polymerisation leads to lamellipodium extension and membrane ruffles at the leading edge of the cell [55],

inflammation. Modified from Davies [46].

and Cdc42 induces formation of microspikes/filopodia [56].

**Figure 5. Cellular processes occurring during migration.** The transition from a stationary state to a migratory phenotype involves several distinct mechanisms, Rho-like GTPases regulate the majority of these processes and the individual GTPases most directly linked to each are: (1.) Rac-dependent actin (blue) polymerisation leads to lamellipodium extension at the leading edge of the cell, (2.) new ECM adhesions are formed at the lamellipodium via focal contacts (red), also regulated by Rac, (3.) cell body contraction is dependent on actomyosin contraction (blue arrows) and is dependent on Rho activity, (4.) tail detachment is possibly affected by GTPase activity but the protease calpain is important for degradation of focal adhesions at the rear of migrating cells, (5.) cadherin-mediated cell-cell adhesions (green) are dependent on Rho and Rac activity and are often downregulated during migration. MMPmediated ECM degradation is necessary for migration, even in cells migrating over a basement membrane.

Several *in vitro* studies have demonstrated activity of the RhoGTPase family is essential for airway epithelial wound repair; activation of protein kinase A (PKA) facilitates scrape wound closure in cultured bovine airway epithelial cells via inhibition of Rho activity and decreased focal contact attachments [57]. The 16HBE human airway epithelial cell line has been used to demonstrate coordination of RhoA and Rac1 activities are vital for efficient wound closure [58]. Detachment of the trailing edge is often a limiting factor of cellular migration speed, detachment is possibly affected by GTPase activity but the protease calpain is important for degradation of focal adhesions at the rear of migrating cells. Rho-GTPases are also involved in cell proliferation during airway epithelial repair; the scaffold IQ domain GTPase-activating protein (IQGAP1), an effector of Rho GTPases, is upregulated during 16HBE scrape wound repair and facilitates closure by increasing via a ß-cateninmediated transcription of genes involved in cell cycle regulation such as CyclinD1 [59, 60].

#### **3.3. The effect of intracellular protein glycosylation on epithelial repair**

The activity of the Rho-GTPase family is highly regulated by glycosylation. *Clostridium difficile* toxins A and B (TcdA and TcdB) are major virulence factors for the induction of antibiotic-associated diarrhea and pseudomembranous colitis. These toxins possess potent

cytotoxicity against cultured cell lines by inducing disaggregation of the cytoskeleton. TcdA and TcdB cellular toxicity is dependent on their intrinsic glucosyltransferase activity, they modify Rho-like GTPases by using UDP-glucose as a co-substrate [61]. RhoA undergoes mono-O-glucosylation at threonine-37, and Rac1 and Cdc42 are glucosylated at the homologous amino acid threonine-35. The threonine residue at position 37 of Rho (position 35 of Rac or Cdc42) is highly conserved and is located in the so-called effector region, it participates in co-ordination of the bound magnesium ion and thereby plays a pivotal role in nucleotide binding [62]. Glucose binding to residues within the effector region inhibits Rho-GTPase activity leading to drastic alteration of the cell cytoskeleton via several potential mechanisms including; inhibition of effector coupling, inhibition of nucleotide exchange (thereby directly blocking GTPase activity), by altering the sub-cellular localization of the GTPase, and finally by altering the stability of the enzyme within the cell (Reviewed in [52], [63]). TcdA has also been shown to induce apoptosis of intestinal epithelial cells via activation of caspases-3, -8 and -9 [64]. To date there is no direct experimental evidence linking Rho-GTPase glucosylation to the inhibition of wound repair in airway epithelial cells. However as this family of enzymes play such a fundamental role in repair of the airway epithelium, and the structure and function of the Rho-family of GTPases is very highly conserved, it is logical to presume that glycosylation would also play a pivotal role in regulating GTPase activity and thus wound repair.

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 201

surface of normal and migrating corneal epithelium of the rat. In their study they found that cell-surface of migrating epithelia express different sugar moieties relative to cell membranes of stratified stationary epithelia. There was a dramatic increase in concanavalin A (ConA) and wheat germ agglutinin (WGA) binding on migrating cells relative to stationary cells. The authors also showed that migrating cells have an increase in glycoprotein production determined by an increase in the incorporation of radiolabelled leucine and glucosamine. In addition they found that N-glycosylation of epithelial cells is necessary for epithelial cell migration [79, 80]. Sweatt *et al*. demonstrated an increase in Nacetylgalactosamine in cell-surface glycoconjugates at the site of epithelial injury in pig cornea [81]. Dorscheid *et al*. has previously characterized cell-surface glycosylation in non-secretory cells of central human airway epithelium and airway epithelial cell lines utilizing lectinbinding patterns [82]. In this study it was shown that galactose- or galactosamine-specific lectins labeled basal epithelial cells and cell lines derived from basal cells. Lectins specific for several different carbohydrate structures bound columnar epithelial cells, and certain fucosespecific lectins labeled subsets of the airway epithelial cells. The cellular specificity of these differences suggests they may be relevant in various cellular functions. It has also been demonstrated that following mechanical injury of guinea pig tracheal epithelium, glycosylation profiles in the repairing epithelium change over time [83]. These changes may represent either the expression of one or more new glycoproteins, or changes in glycosylation of constitutive proteins, required for activation or a change in cell function needed for repair to proceed. Studies have shown that injury of the respiratory epithelium enhances *P. aeruginosa* adhesion and it has been speculated that changes of cell surface glycoconjugates related to

wound repair, cell migration and/or spreading may favor *P. aeruginosa* adhesion [84].

carbohydrates in mediating airway epithelial repair.

In a previous study, Dorscheid *et al*. examined the functional role of cell surface carbohydrates in an *in vitro* model of wound repair after mechanical injury of human airway epithelial cells. The results demonstrated that N-glycosylated glycoproteins, particularly those with a terminal fucose residue, are essential in the adhesion and migration of airway epithelial cells and facilitate closure of epithelial wounds in monolayer culture [85]. Recently, Allahverdian *et al*. studied the role of a fucose containing tetrasaccharide, sialyl-Lewis X (sLex) [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], in airway epithelial repair. Increased presentation of sLex was observed in areas of epithelial damage relative to areas of intact epithelium. In an *in vitro* model of bronchial epithelial repair, cell surface expression of sLex was shown to be significantly increased following mechanical injury of airway epithelial cell monolayers and inhibition of sLex completely prevented repair [86]. Further studies demonstrated that sLex decoration of epidermal growth factor receptor (EGFR) plays an important role in mediating airway epithelial wound repair [87]. Allahverdian *et al*. demonstrated that sLex co-localizes with EGFR and blocking of sLex with a neutralizing antibody resulted in reduced phosphorylation of EGFR and prevented repair. The final step in the synthesis of sLex is catalysed by a specific α-1,3-fucosyltransferase, fucosyltransferase-IV (FucT-IV). Reduction in EGFR phosphorylation and repair were similarly observed when FucT-IV gene expression was knocked down using small interfering RNA (siRNA). These studies demonstrate the importance of cell surface

## **3.4. Role of cell surface glycoconjugates in epithelial repair**

Many proteins essential for normal cell physiology including adhesion molecules and cell surface receptors are glycosylated [65,66]. Alteration in the glycosylation pattern of many glycoproteins leads to changes in their function. It has been shown that impaired glycosylation of receptors often leads to abnormal intracellular trafficking, ligand binding and downstream signal transduction ability [67-70]. Tsuda *et al*. demonstrated that removal of sialic acid from erythropoietin leads to loss of its *in vivo* activity [71]. Complex carbohydrate structures attached to cell surface proteins and lipids have functional roles in cell motility [72], adhesion [73], proliferation [74], and growth potential [75] in several cell types. It also has been demonstrated that certain apical cell-surface carbohydrates are altered during cellular differentiation [76]. Thus many cellular functions are regulated and dependent upon glycoproteins. Because of the role of glycosylated structures in cell-cell and cell-matrix interaction, it is not surprising that there is a growing interest to explore the role for and regulation of these cell-surface carbohydrates in epithelial repair.

Lectins are naturally occurring proteins that can be isolated from a variety of plants and animals. Each lectin binds to a specific sugar moiety. As such, lectins are exquisitely selective tools to identify or block specific glycoconjugate motifs and have been extensively employed to study the role of cell-surface sugars and complex carbohydrates in cellular function [77, 78]. A variety of approaches have been employed to unravel the role(s) of carbohydrates in the multiple steps of the repair process. Some studies have investigated the expression pattern of different carbohydrates after injury using lectins as probes. Using three methods for localizing or quantifying lectin-binding sites Gipson *et al*. compared cellsurface of normal and migrating corneal epithelium of the rat. In their study they found that cell-surface of migrating epithelia express different sugar moieties relative to cell membranes of stratified stationary epithelia. There was a dramatic increase in concanavalin A (ConA) and wheat germ agglutinin (WGA) binding on migrating cells relative to stationary cells. The authors also showed that migrating cells have an increase in glycoprotein production determined by an increase in the incorporation of radiolabelled leucine and glucosamine. In addition they found that N-glycosylation of epithelial cells is necessary for epithelial cell migration [79, 80]. Sweatt *et al*. demonstrated an increase in Nacetylgalactosamine in cell-surface glycoconjugates at the site of epithelial injury in pig cornea [81]. Dorscheid *et al*. has previously characterized cell-surface glycosylation in non-secretory cells of central human airway epithelium and airway epithelial cell lines utilizing lectinbinding patterns [82]. In this study it was shown that galactose- or galactosamine-specific lectins labeled basal epithelial cells and cell lines derived from basal cells. Lectins specific for several different carbohydrate structures bound columnar epithelial cells, and certain fucosespecific lectins labeled subsets of the airway epithelial cells. The cellular specificity of these differences suggests they may be relevant in various cellular functions. It has also been demonstrated that following mechanical injury of guinea pig tracheal epithelium, glycosylation profiles in the repairing epithelium change over time [83]. These changes may represent either the expression of one or more new glycoproteins, or changes in glycosylation of constitutive proteins, required for activation or a change in cell function needed for repair to proceed. Studies have shown that injury of the respiratory epithelium enhances *P. aeruginosa* adhesion and it has been speculated that changes of cell surface glycoconjugates related to wound repair, cell migration and/or spreading may favor *P. aeruginosa* adhesion [84].

200 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

a pivotal role in regulating GTPase activity and thus wound repair.

**3.4. Role of cell surface glycoconjugates in epithelial repair**

for and regulation of these cell-surface carbohydrates in epithelial repair.

Many proteins essential for normal cell physiology including adhesion molecules and cell surface receptors are glycosylated [65,66]. Alteration in the glycosylation pattern of many glycoproteins leads to changes in their function. It has been shown that impaired glycosylation of receptors often leads to abnormal intracellular trafficking, ligand binding and downstream signal transduction ability [67-70]. Tsuda *et al*. demonstrated that removal of sialic acid from erythropoietin leads to loss of its *in vivo* activity [71]. Complex carbohydrate structures attached to cell surface proteins and lipids have functional roles in cell motility [72], adhesion [73], proliferation [74], and growth potential [75] in several cell types. It also has been demonstrated that certain apical cell-surface carbohydrates are altered during cellular differentiation [76]. Thus many cellular functions are regulated and dependent upon glycoproteins. Because of the role of glycosylated structures in cell-cell and cell-matrix interaction, it is not surprising that there is a growing interest to explore the role

Lectins are naturally occurring proteins that can be isolated from a variety of plants and animals. Each lectin binds to a specific sugar moiety. As such, lectins are exquisitely selective tools to identify or block specific glycoconjugate motifs and have been extensively employed to study the role of cell-surface sugars and complex carbohydrates in cellular function [77, 78]. A variety of approaches have been employed to unravel the role(s) of carbohydrates in the multiple steps of the repair process. Some studies have investigated the expression pattern of different carbohydrates after injury using lectins as probes. Using three methods for localizing or quantifying lectin-binding sites Gipson *et al*. compared cell-

cytotoxicity against cultured cell lines by inducing disaggregation of the cytoskeleton. TcdA and TcdB cellular toxicity is dependent on their intrinsic glucosyltransferase activity, they modify Rho-like GTPases by using UDP-glucose as a co-substrate [61]. RhoA undergoes mono-O-glucosylation at threonine-37, and Rac1 and Cdc42 are glucosylated at the homologous amino acid threonine-35. The threonine residue at position 37 of Rho (position 35 of Rac or Cdc42) is highly conserved and is located in the so-called effector region, it participates in co-ordination of the bound magnesium ion and thereby plays a pivotal role in nucleotide binding [62]. Glucose binding to residues within the effector region inhibits Rho-GTPase activity leading to drastic alteration of the cell cytoskeleton via several potential mechanisms including; inhibition of effector coupling, inhibition of nucleotide exchange (thereby directly blocking GTPase activity), by altering the sub-cellular localization of the GTPase, and finally by altering the stability of the enzyme within the cell (Reviewed in [52], [63]). TcdA has also been shown to induce apoptosis of intestinal epithelial cells via activation of caspases-3, -8 and -9 [64]. To date there is no direct experimental evidence linking Rho-GTPase glucosylation to the inhibition of wound repair in airway epithelial cells. However as this family of enzymes play such a fundamental role in repair of the airway epithelium, and the structure and function of the Rho-family of GTPases is very highly conserved, it is logical to presume that glycosylation would also play

> In a previous study, Dorscheid *et al*. examined the functional role of cell surface carbohydrates in an *in vitro* model of wound repair after mechanical injury of human airway epithelial cells. The results demonstrated that N-glycosylated glycoproteins, particularly those with a terminal fucose residue, are essential in the adhesion and migration of airway epithelial cells and facilitate closure of epithelial wounds in monolayer culture [85]. Recently, Allahverdian *et al*. studied the role of a fucose containing tetrasaccharide, sialyl-Lewis X (sLex) [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], in airway epithelial repair. Increased presentation of sLex was observed in areas of epithelial damage relative to areas of intact epithelium. In an *in vitro* model of bronchial epithelial repair, cell surface expression of sLex was shown to be significantly increased following mechanical injury of airway epithelial cell monolayers and inhibition of sLex completely prevented repair [86]. Further studies demonstrated that sLex decoration of epidermal growth factor receptor (EGFR) plays an important role in mediating airway epithelial wound repair [87]. Allahverdian *et al*. demonstrated that sLex co-localizes with EGFR and blocking of sLex with a neutralizing antibody resulted in reduced phosphorylation of EGFR and prevented repair. The final step in the synthesis of sLex is catalysed by a specific α-1,3-fucosyltransferase, fucosyltransferase-IV (FucT-IV). Reduction in EGFR phosphorylation and repair were similarly observed when FucT-IV gene expression was knocked down using small interfering RNA (siRNA). These studies demonstrate the importance of cell surface carbohydrates in mediating airway epithelial repair.

It has been shown previously by Donaldson *et al*. that the plant lectin concanavalin A (ConA), which binds glucose and mannose moieties, can inhibit migration of newt epidermal cells [88]. Gipson *et al*. cultured rat corneas with 3 mm central epithelial abrasions in the presence of four plant lectins. In this study the authors demonstrated that blocking glucose, mannose, and glucosamine sites on corneal epithelial cell surfaces and/ or the epithelial basement membrane reversibly slows or inhibits epithelial migration [78]. Using the same culture model of bronchial epithelial repair, Patchell *et al*. has demonstrated that following mechanical wounding of intact monolayers, the lectins *Allomyrina dichotoma* (AlloA) and chick pea agglutinin (CPA) differentially stain human airway epithelial cells in damaged areas relative to the staining of intact epithelial monolayers [89]. While AlloA positive staining cells are those that appear to be migrating from areas distant to the wound and accumulating in the wound, CPA positive staining cells are restricted to the leading edge of the wound. Moreover, the addition of the above lectins following mechanical wounding inhibited the repair. These results suggest that AlloA and CPA bind specific carbohydrate structures involved in normal epithelial repair. Further work is being carried out to determine the identity of the relevant proteins associated with these carbohydrate ligands.

IL-13, Asthma and Glycosylation in Airway Epithelial Repair 203

date, as many as 14 galectins have been identified, and they have been found in nonmammalian species such as birds, amphibians, fish, worms, sponges and fungi. The binding affinities of galectins are often greater for the oligosaccharides than the monosaccharide galactose. The expression of galectins is conserved however; their expression is often tissue specific and developmentally regulated. Different cells express a unique complement of galectins. Of the 14 galectins, eight have been identified in the nucleus and cytoplasm and participate in specific cellular functions. Nine galectins have been found extracellularly. Their presentation on the cell surface remains a mystery since they lack a signal sequence for

Studies have shown that galectin-1 and galectin-3 have the potential to mediate cell-matrix interactions [92-95]. Their ubiquitous expression makes it difficult to understand the mechanism of their involvement in this process. In corneal epithelial cells, galectin-3 and galectin-7 have been characterized as mediators of epithelial cell migration, an essential component of epithelial wound repair [96, 97]. Galectin immunostaining in healing wounds is more intense relative to normal epithelium, primarily at the leading edge and at areas of cell-matrix interactions [97]. Furthermore, wound repair was impaired in gal-3-/- mice in the wound model systems of either alkali burn wounds that leave the corneal stroma intact or laser ablation wounds that result in damage to the underlying basement membrane. This impaired repair was not seen in gal-1-/- mice. The authors propose that this effect is mediated by the lectin domain binding complementary glycoconjugates in the ECM and cell surface molecules resulting in enhanced cell-cell and cell-matrix interactions [97]. Similar work has demonstrated that galectin-7 also plays a role in corneal epithelial repair [96]. Although obvious differences exist between tissues, galectins may play a similar role in other

Annexins are a family of proteins that bind acidic phospholipids in a calcium dependant manner. Similar to galectins, annexins are largely cytoplasmic, however; several annexins have been detected on the cell surface of a variety of cells. Like galectins, annexins also lack a signal sequence for the cell surface presentation [98]. Annexins are proteins that have been associated with many cellular functions however their role remains poorly understood. Recent work has suggested that annexins possess carbohydrate binding abilities. This was first observed with annexin A4 when it was shown that it binds a variety of glycans [99]. Recent studies have suggested that annexin II (AII) is also capable of binding carbohydrates however this work is still in preliminary stages. Previously, the binding of tissue-type plasminogen activator (t-PA) was shown to bind HepG2 cells, however, following enzymatic removal of α-fucose residues on t-PA, this binding was dramatically decreased [100]. Subsequently, AII has been shown to be a cell surface receptor for t-PA [101]. The importance of α-fucose on t-PA may be a result of the carbohydrate binding of AII. Patchell *et al.* has shown that AII is presented on the cell surface and acts as a mediator of epithelial repair. AII was shown to be actively involved in wound repair in cells at the leading edge of the wound. Interestingly, the initial isolation purification of AII was achieved using a lectin, chick pea agglutinin, suggesting that itself, or an associated protein is glycosylated. Their tight association with the membrane and potential carbohydrate binding may influence the

secretion via the classical secretory pathway for membrane proteins.

epithelial organs and epithelial repair such as the lung and intestinal tract.