Evolution of anti-modified protein antibody responses can be driven by consecutive exposure to different post-translational modifications

Abstract

Background: Besides anti-citrullinated protein antibodies (ACPA), rheumatoid arthritis patients (RA) often display autoantibody reactivities against other post-translationally modified (PTM) proteins, more specifically carbamylated and acetylated proteins. Immunizing mice with one particular PTM results in an anti-modified protein antibody (AMPA) response recognizing different PTM-antigens. Furthermore, human AMPA, isolated based on their reactivity to one PTM, cross-react with other PTMs. However, it is unclear whether the AMPA-reactivity profile is "fixed" in time or whether consecutive exposure to different PTMs can shape the evolving AMPA response towards a particular PTM.

Methods: Longitudinally collected serum samples of 8 human individuals at risk of RA and 5 with early RA were tested with ELISA, and titers were analyzed to investigate the evolution of the AMPA responses over time. Mice (13 per immunization group in total) were immunized with acetylated (or carbamylated) protein (ovalbumin) twice or cross-immunized with an acetylated and then a carbamylated protein (or vice versa) and their serum was analyzed for AMPA responses.

Results: Human data illustrated dynamic changes in AMPA-reactivity profiles in both individuals at risk of RA and in early RA patients. Mice immunized with either solely acetylated or carbamylated ovalbumin (AcOVA or CaOVA) developed reactivity against both acetylated and carbamylated antigens. Irrespective of the PTM-antigen used for the first immunization, a booster immunization with an antigen bearing the other PTM resulted in increased titers to the second/booster PTM. Furthermore, cross-immunization skewed the overall AMPA-response profile towards a relatively higher reactivity against the "booster" PTM.

Conclusions: The relationship between different reactivities within the AMPA response is dynamic. The initial exposure to a PTM-antigen induces cross-reactive responses that can be boosted by an antigen bearing this or other PTMs, indicating the formation of cross-reactive immunological memory. Upon subsequent exposure to an antigen bearing another type of PTM, the overall reactivity pattern can be skewed towards better recognition of the later encountered PTM. These data might explain temporal differences in the AMPA-response profile and point to the possibility that the PTM responsible for the initiation of the AMPA response may differ from the PTM predominantly recognized later in time.

Keywords: (AAPA) Anti-modified protein antibodies (AMPA); Anti-acetylated protein antibodies; Anti-carbamylated protein antibodies (Anti-CarP); Anti-citrullinated protein antibodies (ACPA); Rheumatoid arthritis.

Fig. 1

The shift of the AMPA reactivity in individuals at risk of RA and RA patients. A, B Different AMPA (targeting citrullinated, carbamylated or acetylated antibodies) titers as calculated by ELISA with modified fibrinogen. Human serum samples collected at different timepoints were measured. The data are depicted as clustered columns to simplify comparison of each AMPA reactivity between the timepoints, different PTM-reactivities should not be compared directly. The difference between the timepoints was 3 years for individuals at risk and 4 months for RA patients. Shown are inverted AMPA titers of 8 individuals at risk of RA and 5 RA patients

Fig. 2

Evolution of the AMPA-reactivity profile in mice immunized with different PTM-antigens. A ELISA of the titrated murine sera showing the development of anti-modified protein antibody (AMPA) response to different post-translational modifications of fibrinogen, namely acetylated (AcFib) and carbamylated (CaFib) variants. Individual graphs depict titration ELISA results measured in the end of the experiment in mice groups with different immunization strategies. Medians and interquartile intervals per serum dilution are shown. Immunization experiments were performed twice with similar results. Data from one of the two representative immunization experiments are shown. B Timelines demonstrating median anti-AcFib and anti-CaFib reactivity of the immunized mice per immunization group. Arrows indicate the timepoints at which the mice were immunized (week 0 and week 5). Graphs depict ELISA OD-s measured with sera collected at different timepoints and diluted 1:50 for ELISA; medians and interquartile intervals are shown per timepoint. Data from one of the two representative immunization experiments are shown. p values (asterisk) refer to the change between two immunization groups within one timepoint according to Mann-Whitney U test (*p < 0.05). C Changes in AMPA titers of individual mice within 1 week after the booster (between timepoints 5 and 6), calculated by subtraction of the log-transformed timepoint 5 titer from the timepoint 6 titer; means ± SD are shown per immunization group. Pooled titer data from two immunization experiments are shown. p values (asterisk) refer to the change between two immunization groups according to Mann-Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001). All experiments were performed twice with similar results. The data obtained from one experiment is shown. D Relative evolution of the anti-CarP and AAPA responses: the titer evolution data were summarized per immunization group by depicting average titers. Due to the exponential nature of titer values, geometric mean was used to determine the average. Pooled titer data from the two immunization experiments are shown, measured with serum collected at week 5, week 6, and week 9. Example of performed calculations is shown in supplementary methods. E Model illustrating multireactive nature of AMPA is shown together with which antibodies are measured in AMPA assays applied in the study