Retinoic acid enhances lactoferrin-induced IgA responses by increasing betaglycan expression

Cellular & Molecular Immunology (2016) 13, 862–870 ß 2016 CSI and USTC. All rights reserved 1672-7681/16 $32.00 www.nature.com/cmi RESEARCH ARTICLE Retinoic acid enhances lactoferrin-induced IgA responses by increasing betaglycan expression Jeong-Min Lee1,6, Young-Saeng Jang1,6, Bo-Ra Jin1,6, Sun-Jin Kim1, Hyeon-Jin Kim1, Bo-Eun Kwon2, Hyun-Jeong Ko2, Sung-il Yoon3, Geun-Shik Lee4, Woan-Sub Kim5, Goo-Young Seo1 and Pyeung-Hyeun Kim1 Lactoferrin (LF) and retinoic acid (RA) are enriched in colostrum, milk, and mucosal tissues. We recently showed that LF-induced IgA class switching through binding to betaglycan (transforming growth factor-beta receptor III, TbRIII) and activation of canonical TGF-b signaling. We investigated the combined effect of LF and RA on the overall IgA response. An increase in IgA production by LF was further augmented by RA. This combination effect was also evident in Ig germ-line a (GLa) transcription and GLa promoter activity, indicating that LF in cooperation with RA increased IgA isotype switching. We subsequently found that RA enhanced TbRIII expression and that this increase contributed to LF-stimulated IgA production. In addition to the IgA response, LF and RA in combination also enhanced the expression of the gut-homing molecules C-C chemokine receptor 9 (CCR9) and a4b7 on B cells. Finally, peroral administration of LF and RA enhanced the frequency of CCR91IgA1 plasma cells in the lamina propria. Taken together, these results suggest that LF in cooperation with RA can contribute to the establishment of gut IgA responses. Cellular & Molecular Immunology (2016) 13, 862–870;doi:10.1038/cmi.2015.73;published online 17 August 2015 Keywords: gut homing molecule; IgA; lactoferrin; retinoic acid; TGF-b receptor INTRODUCTION IgA is the most important antibody that protects mucosal surfaces against pathogens. Numerous IgA-producing B cells are found in the gut lamina propria (LP).1 Gut-tropic B cells robustly express C-C chemokine receptor 9 (CCR9) and a4b7, which play a role in the mucosal trafficking of these cells.2–4 Ig class switch recombination (CSR) allows the recombined variable region gene segment (VDJ) to be expressed with a new downstream heavy chain constant region (CH) gene. CSR is achieved by deletional recombination between switch (S) region sequences placed upstream of each of the CH genes, with the exception of Cd.5 CSR is directed to a particular CH gene by cytokines that stimulate transcription from germ-line (GL) CH genes prior to CSR to the same CH gene. It is well established that transforming growth factor-beta 1 (TGF-b1) induces IgA CSR.6–8 Retinoic acid (RA) is a natural bioactive metabolite of vitamin A that regulates a broad range of biological processes by binding to specific nuclear retinoid receptors.9 Moreover, RA can cause IgA isotype switching,10,11 and GALT-DC-derived RA enhances the expression of the gut-homing molecules CCR9 and a4b7 on B cells.12 We recently demonstrated that RA selectively activated IgA isotype switching and synergized with TGF-b1 to promote IgA production and the expression of gut-homing molecules in mice.13 Lactoferrin (LF) is found at high concentrations on many mucosal surfaces.14 LF induces the activation, proliferation, and differentiation of immune cells. This activity has been related 1 Department of Molecular Bioscience, School of Biomedical Science, Kangwon National University, Chuncheon, Republic of Korea; 2Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University, Chuncheon, Republic of Korea; 3Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Republic of Korea; 4College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea and 5Department of Animal Life and Environmental Science, College of Agriculture and Life Science, Hankyong National University, Anseong-si 456-749, Republic of Korea 6 These authors contributed equally to this work. Correspondence: P-H Kim, Department of Molecular Bioscience, School of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea. E-mail: G-Y Seo, Department of Molecular Bioscience, School of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea. E-mail: Received: 1 April 2015; Revised: 23 June 2015; Accepted: 23 June 2015 Retinoid increases lactoferrin-induced IgA responses J-M Lee et al 863 to a direct effect of LF on the cells through specific LF receptors, including LF receptor (LFR), low-density lipoprotein receptorrelated protein (LRP)-1, nucleolin, and proteoglycan.15,16 Among these receptors, betaglycan (TGFbRIII, TbRIII) is a type of proteoglycan that was originally identified as a nonsignaling coreceptor in TGF-b signaling.17,18 We recently showed that LF caused IgA and IgG2b isotype switching by binding to TbRIII and activating canonical TGF-b signaling.19 Although LF and RA are abundant inmucosal secretions and are known to cause IgA isotype switching, their combined effect on the overall gut IgA response has not been characterized. In this study, we found that LF and RA synergized to enhance IgA isotype switching and the expression of gut-homing molecules on B cells. Moreover, RA increased the expression of TbRIII, which was the LFR and resulted in a robust IgA response. METHODS Animals BALB/c mice were obtained from DaehanBiolink. Co. Ltd (Chungcheongbuk-do, Korea). The animals were fed Purina Laboratory Rodent Chow 5001 ad libitum. Mice that were 8–12 weeks of age were used in this study. Animal care was performed in accordance with the institutional guidelines set forth by Kangwon National University. Cell preparation and reagents Mouse splenic B-cell suspensions were prepared as previously described.7 Briefly, T cells were depleted from the cell suspensions by treatment with a cocktail of anti-Thy1.2, anti-Lyt2.2, and anti-L3T4 monoclonal antibodies and low-tox rabbit complement (Cedarlane, Ontario, Canada). As a result, B cells constituted more than 90% of the residual population as assessed by the presence of surface Ig using flow cytometric analysis. Subsequently, resting splenic CD432 B cells were negatively sorted using 10 mL of anti-CD43 (Ly-48) microbeads per 107 cells and a magnetic column (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. The murine B-cell lymphoma cell line CH12F3-2A (surface m1) was provided by Dr. T. Honjo (Kyoto University, Kyoto, Japan). Bovine LF was supplied by Morinaga Milk Co., Ltd. (Zama, Japan); the preparation contained less than 5.0 pg mg21 of lipopolysaccharide (LPS) (endotoxin).20 Anti-bovine LF antiserum was purchased from Bethyl Laboratories, Inc. (Montgomery, TX, USA). RA and LPS (Escherichia coli O111:B4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TGF-b1 and the antiTGFbRIII antibody (Ab) were purcha (...truncated)