FERNANDEZ Pablo Mariano
congresos y reuniones científicas
Characterization of cytokine-producing B cell subsets in human tonsils, insights into their role in tonsillar hypertrophy and recurrent tonsilitis
Congreso; XII Congress of the Latin American Association of Immunology & XXIII Congress of the Mexican Society of Immunology; 2018
Tonsils are secondary lymphoid organs which must grant immunologic protection against pathogens and tolerance to harmless antigens (Ags) in air and food. They are mostly B-cell (Bc) maturation and differentiation sites. Bc have traditionally been associated with antibody production and Ag presentation. Lately, the Bc field has been transfigured by the attribution of new functions. For instance, Bc secrete multiple cytokines under different stimuli. It is likely that alteration of the immune equilibrium activation/regulation maytrigger recurrent tonsillitis (RT) and/or hypertrophy (HT) leading in turn to tonsillectomy. Hence, the aim of our work was to characterize those unconventional immune functions of tonsillar Bc, i.e different Bc subsets with regulatory and pro-inflammatory profiles, and their association with the disease that led to surgery. Methodology used involved FACS and real time PCR. Initially,we determined tonsillar Bc expression of IL17, IL6, IL8, IFNg,IL5, TNFa, IL10 and TGFb for different patients upon three sets of stimuli (IL2+IL4/IL2+IL4+CpG+CD40L/ aIgM+CD40L). Moreover, we established the kinetics of appearance of IL10 (regulatory cytokine) expressing Bc (Breg) and compared it with that of pro-inflammatory cytokine-expressing Bc (IL6 and IL8) for 5 patients upon 0, 16, 32 and 72 hs stimulation, via TLR9 and CD40. The proportion of Breg increased between 16 and 72 hs in all cases. Notably, the percentage of Breg detected at the different time points depended on the cause of surgery. The hypertrophied (HT) tonsils´ samples showed a significantly (p<0.05) lower percentage of Bregs (4.4±0.4) compared to those from recurrent tonsillitis (RT, 9.9±1.5) as well as a significantly (p<0.05) higher proportion of the germinal center (GC) population (23.8±4.3) than those from children with RT(7.9±1.8). A comparative increment of the GC percentage was not accompanied by aproportional growth of the memory B cell population in those HT samples. In contrast, these samples displayed a significantly (p<0.05) lower percentage of the eBm5 subset (7.8±0.5) than RT samples (15.5±2.1), indicative of a putative blockade between the GC and memory B cell stages on HT patients. Collectively, our results demonstrate that a defective tonsillar Breg compartment indicates an increase in the proportion of GC in vivo and therefore unrestrained T follicular helper (Tfh) function, suggesting that Tfh are atarget population of Breg function. Interestingly, Breg modulated cytokine intracellular expression and proliferation of stimulated CD4+ cells, in co-cultures performed with those autologous sorted subsets at 3 different ratios. Finally, we found Bregs at every stage of B cell diferentiation represented in tonsils, that is, Bregs resulted diffusely scattered throughout the B cell lineage. Our findings provide greater insight into the role of Bregs inGC reactions and characterize for the first time their involvement in the pathogenesis of tonsillar dysfunction. Moreover, we noticed a relevant proportion of IL5 and IL17-expressing B cells in tonsils which is, to our knowledge, the first time that is reported in literature. We consider this work holds relevant implications for basic immunology as well as for translational medicine. In terms of basic immunology, human B cells with regulatoryfunction remain poorly understood. Our results shed some light on the controversy of their phenotype and support the notion that they are indeed a separate lineage, in addition to confirm their novel immune function at the GC reaction. As for translational medicine, there is a clear need for more extensive immunological studies focusing on palatine tonsils. It is unfortunate that their immunologic role is not yet completely understood, in particular having in mind not only that infectious and inflammatory diseases involving the pharynx, tonsils, and adenoids account for a significant proportion of childhood illnesses and pediatric health care expenditures, but also that the oral/nasal route for vaccine administration has proved highly relevant to stimulate both mucosal and systemic immunity.AcknowledgementsThis research was funded through grants from the following Argentinean governmental agencies: ANPCyT (BID PICT 2015-0113) and UBA (20020150200029BA), grants to E.A.L.S.V. received a CONICET postgraduate scholarship. The authors would like to thank the anonymous patients and/or their parents that provided consent for the donation of samples to this research project