Exosomes from lung and breast milk – regulators of immune responses

Exosomes are key mediators of intercellular communication with the capacity to regulate immune responses, locally and distally. Most knowledge on exosomal function has been gained from exosomes generated from in vitro cell cultures, which has helped define the role of exosomes from specific cell types. However, studying ex-vivo isolated exosomes from body fluids are more likely to yield clues about their in vivo role. This thesis aimed to gain knowledge regarding the role of humanderived exosomes from two important organs with a well-equipped immune system, namely the lung and the mammary gland. In study I we studied exosomes from bronchoalveolar lavage fluid (BALF) of patients with pulmonary sarcoidosis and healthy individuals. We detected elevated levels of exosomes with an altered exosome protein profile in BALF from patients compared to exosomes from healthy controls. Exosomes isolated from patients exhibited proinflammatory functions seen by their ability to induce inflammatory cytokine release (IFNγ and IL-13) in autologous peripheral blood mononuclear cells (PBMCs) and (IL8) in the bronchial epithelial cell line (BECs) 16HB14. These data suggest that exosomes may contribute to the inflammatory state of sarcoidosis. In order to investigate the specificity of the findings in study I relative to other inflammatory settings, we performed analysis of BALF exosomes from asthmatics. In study II we found that several surface proteins (CD36, CD63 and CD81) were upregulated on BALF exosomes from asthmatics during steady state and after allergen challenge compared to healthy controls. In addition, BALF-derived exosomes from asthmatics at steady state and after allergen challenge could stimulate secretion of leukotrienes (LTs) and IL-8 in BECs, suggesting a proinflammatory function of BALF exosomes in asthmatic inflammation. Breastfeeding is associated with health benefits for the child with possible effects on allergy development. Variations in immune-composition in breast milk between mothers could potentially result in differences in allergic outcome. Therefore, in study III we sought to determine whether maternal sensitization and lifestyle could affect exosome composition in breast milk. Accordingly, we found that both maternal immune status and environmental factors have a differential impact on subpopulations of exosomes in breast milk with potential effects on allergic outcome of the child as a consequence. In study IV we aimed to study the effect of breast milk-exosomes on HIV infection based on findings suggesting a protective role of breastfeeding on HIV infection from mother-to-child. We report a protective role of human breast milk exosomes on HIV infection of dendritic cells and subsequent transfer and infectivity of T cells. Thus, we suggest that breast milk-exosomes might constitute one protective factor in milk against HIV infection in the child. In conclusion, this thesis provides important insights into the composition and function of exosomes from human BALF and breast milk. Furthermore, it sheds light on how different immunological conditions can perturb the function and phenotype of exosomes in vivo. In the future, this could have important implications for the development of novel treatments against inflammatory diseases using exosomes as targets or therapeutic vehicles. LIST OF PUBLICATIONS I. Qazi KR*, Torregrosa Paredes P*, Dahlberg B, Grunewald J, Eklund A & Gabrielsson S. Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis. Thorax. 2010 Nov;65(11):1016-24 *These authors contributed equally II. Torregrosa Paredes P, Esser J, Admyre C, Nord M, Qazi KR, Lukic A, Radmark O, Gronneberg R, Grunewald J, Eklund A, Scheynius A & Gabrielsson S. Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy. 2012 Jul;67(7):911-9 III. Torregrosa Paredes P*, Gutzeit C*, Johansson S, Admyre C, Stenius F, Alm J, Scheynius A & Gabrielsson S. Differences in exosome populations in human breast milk in relation to maternal allergic sensitization and lifestyle. Allergy. 2014 Jan 15. doi: 10.1111/all.12357. [Epub ahead of print] *These authors contributed equally IV. Naslund IT, Paquin-Proulx D, Torregrosa Paredes P, Vallhov H, Sandberg KJ & Gabrielsson S. Exosomes from breast milk inhibit HIV-1 infection of dendritic cells and subsequent viral transfer to CD4 T cells. AIDS. 2014 Jan 14;28(2): 171-80 PUBLICATIONS NOT INCLUDED IN THIS THESIS Lasser C, Alikhani SV, Ekstrom K, Eldh M, Torregrosa Paredes P, Bossios A, Sjostrand M, Gabrielsson S, Lotvall J & Valadi H. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med. 2011. Jan 14;9:9 Levanen B, Bhakta NR, Torregrosa Paredes P, Barbeau R, Hiltbrunner S, Pollack JL, Skold M, Svartengren M, Grunewald J, Gabrielsson S, Eklund A, Larsson BM, Woodruff PG, Erle DJ, Wheelock A. Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients. J Allergy Clin Immunol. 2013 Mar;131(3):894-903 Villablanca E, De Calisto J, Torregrosa Paredes, Cassani B, Nguyen D, Gabrielsson S & Mora JR. β7 integrins are required to give rise to intestinal mononuclear phagocytes with tolerogenic potential. Gut. 2013 Sep 12. doi: 10.1136/gutjnl-2013-305386. [Epub ahead of print]