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Trained immunity by citrullinated peptides
We will investigate training abilities of citrullinated proteins in healthy human monocytes, discover citrullinated specific monocyte surface receptors, identify innate immune memory mechanisms in monocytes, and analyze the ongoing transcriptomic and chromatin alterations leading to innate immunity.
Immunological memory is a process of adaptive immune response in which specific T and B memory cells are generated. These cells exert long-term protection against specific pathogens. Recent studies revealed the existence of immunological memory in myeloid cells. For instance, macrophages have been described to be trained by vaccination or an infection and subsequently this result in an enhanced response upon re-challenge with a pathogen unrelated to the first one. This phenomenon was defined as innate immunological memory (trained immunity). It has been hypothesized that in early phases of rheumatoid arthritis (RA) pathogenesis, mucosal bacterial pathogens such as P. gingivalis generate cirullinated peptides that in turn initiate a T cell response to these neo-epitopes and finally result in the release of anti-citrullinated peptide autoantibodies (ACPAs) by B cells. In turn, ACPAs immune complexes can activate monocytes. In RA, together with damage associated molecular patterns (DAMPs), this can lead to chronic inflammation, during which pro-inflammatory cytokines are released. We hypothesize that citrullinated peptides can induce innate immunological memory.
Monocytes will be isolated from healthy donors. Then, RA-relevant pathogenic stimuli such as citrullinated recombinant proteins will be tested for the ability to train healthy donor monocytes using an established in vitro training protocol scheme, in which a second unrelated stimulus is added after five days, i.e. E. coli LPS. Twenty-four hours after the second stimulus, the phenotype (including MHC II expression), release of chemokines / cytokines, metabolic and phagocytic activities will be analyzed. These read-outs are criteria confirming that the cells are trained.
RNA sequencing will analyze the transcriptomic changes and ATAQ-sequencing will reveal chromatin regulatory regions. Bioinformatics analysis of two data sets will identify specific transcription factors that are responsible for the trained properties of citrullinated proteins.
Next, citrullinated proteins will be used to identify the cell surface receptor(s) on the monocytes using the LRC-TriCEPS technology developed by DualsystemsBiotech.
It is hypothesized that RA monocytes are already trained, being activated by the disease process. In this case, they should show an enhanced response to subsequent stimuli. In particular, the response to E. coli LPS, P. gingivalis LPS, and RA-relevant DAMPs (e.g. oS100A4) as second stimuli will be tested at baseline and in the in vitro training protocol using RA peripheral blood, synovial fluid and tissue monocytes / macrophages.
In conclusion, this study will provide mechanistic information about the generation of innate immune memory in monocytes from healthy donors and RA patients. In addition, the project will identify a citrullinated specific receptor on monocytes.
Immunological memory is a process of adaptive immune response in which specific T and B memory cells are generated. These cells exert long-term protection against specific pathogens. Recent studies revealed the existence of immunological memory in myeloid cells. For instance, macrophages have been described to be trained by vaccination or an infection and subsequently this result in an enhanced response upon re-challenge with a pathogen unrelated to the first one. This phenomenon was defined as innate immunological memory (trained immunity). It has been hypothesized that in early phases of rheumatoid arthritis (RA) pathogenesis, mucosal bacterial pathogens such as P. gingivalis generate cirullinated peptides that in turn initiate a T cell response to these neo-epitopes and finally result in the release of anti-citrullinated peptide autoantibodies (ACPAs) by B cells. In turn, ACPAs immune complexes can activate monocytes. In RA, together with damage associated molecular patterns (DAMPs), this can lead to chronic inflammation, during which pro-inflammatory cytokines are released. We hypothesize that citrullinated peptides can induce innate immunological memory. Monocytes will be isolated from healthy donors. Then, RA-relevant pathogenic stimuli such as citrullinated recombinant proteins will be tested for the ability to train healthy donor monocytes using an established in vitro training protocol scheme, in which a second unrelated stimulus is added after five days, i.e. E. coli LPS. Twenty-four hours after the second stimulus, the phenotype (including MHC II expression), release of chemokines / cytokines, metabolic and phagocytic activities will be analyzed. These read-outs are criteria confirming that the cells are trained. RNA sequencing will analyze the transcriptomic changes and ATAQ-sequencing will reveal chromatin regulatory regions. Bioinformatics analysis of two data sets will identify specific transcription factors that are responsible for the trained properties of citrullinated proteins. Next, citrullinated proteins will be used to identify the cell surface receptor(s) on the monocytes using the LRC-TriCEPS technology developed by DualsystemsBiotech. It is hypothesized that RA monocytes are already trained, being activated by the disease process. In this case, they should show an enhanced response to subsequent stimuli. In particular, the response to E. coli LPS, P. gingivalis LPS, and RA-relevant DAMPs (e.g. oS100A4) as second stimuli will be tested at baseline and in the in vitro training protocol using RA peripheral blood, synovial fluid and tissue monocytes / macrophages. In conclusion, this study will provide mechanistic information about the generation of innate immune memory in monocytes from healthy donors and RA patients. In addition, the project will identify a citrullinated specific receptor on monocytes.
Overall, this project will allow the identification of new molecular targets to disrupt innate immune memory in RA and other inflammatory diseases.
Overall, this project will allow the identification of new molecular targets to disrupt innate immune memory in RA and other inflammatory diseases.
Prof. Michel Neidhart, PhD
michel.neidhart@usz.ch
Center of Experimental Rheumatology, Clinic of Rheumatology, University Hospital Zürich, Wagistrasse 14, 8952 Schlieren
Prof. Michel Neidhart, PhD michel.neidhart@usz.ch Center of Experimental Rheumatology, Clinic of Rheumatology, University Hospital Zürich, Wagistrasse 14, 8952 Schlieren