Next, we biochemically characterized the PTMs pre sent on EPRO derived murine NETs with a goal of test ing their immunogenicity in vivo. EPRO derived NETs were prepared as for human cells described earlier and subjected to ionomycin and phorbol myristate Volasertib msds acetate as two additional stimuli. When com paring HL 60 and EPRO derived NETs, the majority of the histone PTMs were consistent during NETosis and also similar in response to diverse stimuli. Specifically, in comparing EPRO derived NETs to corresponding unstimulated neutrophils, we observed an even more striking pattern of PTMs associated with a transcription ally silent state than for HL 60 cells. Furthermore, we observed an increase in the silencing marks di methyl H3K9 and tri methyl H4K20.

In contrast with HL 60 derived NETs, however, we observed a strong citrullina tion signature in NETs prepared from EPRO derived neutrophils, clearly demonstrating abun dant citrullination of histones H3 and H4 and consistent with previous studies and primary human PMNs. Murine NETs are weakly immunogenic in vivo We next tested the immunogenicity of Inhibitors,Modulators,Libraries NETs derived from the murine EPRO cell line in vivo. NETs prepared using hydrogen peroxide stimulation of EPRO cells were subcutaneously injected into two groups Inhibitors,Modulators,Libraries of female BALB c wild type mice weekly over 28 days, with one group receiving NETs alone, and another receiving NETs in combination with CRAMP, the murine analo gue of LL 37. Trace or low levels of urinary protein were measured during the remainder of the time course, suggesting a weak response to the NET immunization.

Inhibitors,Modulators,Libraries Autoantibody profiles reflected Inhibitors,Modulators,Libraries reproducible and time dependent responses for IgG and IgM and targeted diverse auto antigens, although these responses were modest and transient. Reactivity to 28 antigens was observed for both IgG and IgM isotypes, including many components within neutrophils or NETs, such as myeloperoxidase, catalase, histones, as well as single and double stranded DNA. Nonetheless, mice immunized with NETs showed significant differential IgM and IgG reac tivity towards many of the same antigens, as well as others. Furthermore, we observed reproducible IgG seroreactiv ity targeting human IgG in mice immunized with NETs derived in serum free conditions, possibly reflecting a species cross reactive rheumatoid factor activity precipi tated by citrullinated murine NETs.

Taken together, the autoantibody reactivity profiles are consistent with the antigenic components of the NET immunogen but sug gest a Inhibitors,Modulators,Libraries modest response to common lupus autoantigens. Autoreactivity profiles of PTM specific histone pep tides from the HEMP arrays revealed a transient IgG response in one individual mouse that disappeared Sorafenib Tosylate side effects by the third month and very little reactivity in the other two mice. There was little or no reactivity to adjuvanted CRAMP or its human analogue, LL 37.