Cow’s milk protein β-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells
Authors: Frank Redegeld
Zdenek Dvorak
Bart R Blokhuis
Karin Hufnagl
Isabella Pali-Schöll
Alessandro Fiocchi
Erika Jensen-Jarolim
Eva Untersmayr
Sheriene Moussa Afify
Andreas Regner
Luis F Pacios
Franziska Roth-Walter
Lisa-Marie Petje
Journal: Journal of Allergy and Clinical Immunology (2021)
Discipline: IMMUNOLOGY
Keywords: Allergen; BLG; Bos d 5; allergy; cow's milk
Affiliations: The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria; Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: ., The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria; Laboratory Medicine and Immunology Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt., Biotechnology Department, ETSIAAB, Center for Plant Biotechnology and Genomics, CBGP (UPM-INIA), Technical University of Madrid, Madrid, Spain., Faculty of Science, Division of Pharmacology, Department of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands., The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria., Childrens Hospital Bambino Gesù, Rome, Italy., Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria., Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic., The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria; Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Abstract:  Background: Beta-lactoglobulin (BLG) is a bovine lipocalin in milk with an innate defense function. The circumstances under which BLG is associated with tolerance of or allergy to milk are not understood. Objective: Our aims were to assess the capacity of ligand-free apoBLG versus loaded BLG (holoBLG) to protect mice against allergy by using an iron-quercetin complex as an exemplary ligand and to study the molecular mechanisms of this protection. Methods: Binding of iron-quercetin to BLG was modeled and confirmed by spectroscopy and docking calculations. Serum IgE binding to apoBLG and holoBLG in children allergic to milk and children tolerant of milk was assessed. Mice were intranasally treated with apoBLG versus holoBLG and analyzed immunologically after systemic challenge. Aryl hydrocarbon receptor (AhR) activation was evaluated with reporter cells and Cyp1A1 expression. Treated human PBMCs and human mast cells were assessed by fluorescence-activated cell sorting and degranulation, respectively. Results: Modeling predicted masking of major IgE and T-cell epitopes of BLG by ligand binding. In line with this modeling, IgE binding in children allergic to milk was reduced toward holoBLG, which also impaired degranulation of mast cells. In mice, only treatments with holoBLG prevented allergic sensitization and anaphylaxis, while sustaining regulatory T cells. BLG facilitated quercetin-dependent AhR activation and, downstream of AhR, lung Cyp1A1 expression. HoloBLG shuttled iron into monocytic cells and impaired their antigen presentation. Conclusion: The cargo of holoBLG is decisive in preventing allergy in vivo. BLG without cargo acted as an allergen in vivo and further primed human mast cells for degranulation in an antigen-independent fashion. Our data provide a mechanistic explanation why the same proteins can act either as tolerogens or as allergens. 

Retracted: No

PubPeer comments:
 It is the multimillion dollar question what makes an allergen an allergen. Using u00df-lactoglobulin (BLG or Bos d5 in allergen nomenclature) the authors show here that  apo-BLG is pro-allergic and  holo-BLG , a complex with quercetin and iron has anti-allergic properties. I can t comment on the molecular modelling but the empirical data are questionable if the authors do not provide any data on BLG purity. Further dialyzation against deionized water was performed (apou2013Bos domesticus 5). HoloBLG was generated by forming ironu2013quercetin 2 (FeQ2) complexes with quercetin (Sigma 1592409) and iron (iron standard AAS, Sigma 16596) at a ratio 2:1 and adjus ing the pH to 7.2 with 10 mM NaOH and adding apoBLG to a final concentration of 1 mM BLG, 2 mM quercetin, and 1 mM iron. Have both BLG fractions really been dialyzed in a different way? Adjusted to different pH? One fraction combined with deferoxamine and the other with quercetin? As BLG is isolated from cow milk there is a good chance that a plethora of molecules including [prohormones like vitD]() are dissolved from BLG depending on the differential preparation. The methods are also contradictory as the mice were split into groups of 5 to 7 animals and treated 6 times on 2 consecutive days while the Fig 2 legend says mice were intranasally sensitized 6 times at biweekly intervals FeQ2 and deferoxamine are used in control groups, but there is no animal group that is treated with quercetin alone. This is an unfortunate omission as quercetin may have [anti-inflammatory properties](www.doi.org 10.3390 nu8030167) itself (and is even able of Ahr activation as seen in Fig 4B). The clinical data are also questionable. If values are standardized against a comparison group, the significance cannot be tested as shown below. A 20% lower IgE binding isn t also unexpected when using a chemically modified allergen (see above). There is also no indication that a 0-20% lower IgE binding capacity is clinically relevant as we are dealing here with a potent allergen that even elicits a reaction in trace amounts. ![file] As far as I see, no other working group replicated the experimental findings although the BLG story is now around for nearly 10 years. Irrespective of that [holo BLG is now patented by the authors] and [sold as a treatment for allergy] . , 
 **I am happy to respond to the queries:** #1:  I can t comment on the molecular modelling but the empirical data are questionable if the authors do not provide any data on BLG purity.  As far as I see, no other working group replicated the experimental findings although the BLG story is now around for nearly 10 years. **We prepared the BLG-stock as apo (for apo and holo) before loading with iron-quercetin. As such, the purity of apo- and holo- was the same and still showed these marked differences. Indeed, everybody can replicate these data and indeed we established and use these standardized conditions for multiple allergens (Betv1, Alta1, BLG Bosd5) and as such are confident that everybody can and will replicate these data. ** #2 The methods are also contradictory as the mice were split into groups of 5 to 7 animals and treated 6 times on 2 consecutive days while the Fig 2 legend says mice were intranasally sensitized 6 times at biweekly intervals **Methods are not contradictory with Fig2A depicting the immunization protocol, showing two days nasal sensitization, then two weeks later, again 2days sensitization and so on for in total 6 times** #3 FeQ2 and deferoxamine are used in control groups, but there is no animal group that is treated with quercetin alone. This is an unfortunate omission as quercetin may have anti-inflammatory properties itself (and is even able of Ahr activation as seen in Fig 4B). **We completely agree that quercetin alone has also anti-inflammatory properties, but as seen in the supplementary data and from our pre-animal experiments, alone it further supports a Th2 environment.** #3 The clinical data are also questionable. If values are standardized against a comparison group, the significance cannot be tested as shown below. A 20% lower IgE binding isn t also unexpected when using a chemically modified allergen (see above). There is also no indication that a 0-20% lower IgE binding capacity is clinically relevant as we are dealing here with a potent allergen that even elicits a reaction in trace amounts. **As can be seen, we used the same sera for both apo- and holo-BLG of children allergic to milk and patients tolerizing milk and thus have an internal control. As can be read in the discussion section, we do not claim that the holo-version does not cause clinically reactivity, but it reveals a very important aspect on the etiology , as it emphasizes that during sensitization at least partly the antigen was presented in its apo-form as otherwise no antibodies against the specific epitopes could have been generated.** , 
 #1 To the comments, I would like to quote the emperor Trajan to Plinius as it summarize perfectly my thoughts on anonymous writings, which is  nam et pessimi exempli nec nostri saeculi est . , 
  argumentum ad hominem non punctum ... [PubPeer follows a good tradition here] Because reviewing is a reciprocal process, meant to be a safeguarding space for valid scientific input, anonymous reviewing is the standard default option. Anonymity should mean protection for the reviewer from retaliation and discrimination. ,