Determining the impact of macrophage-derived Dnase1L3 in lupus-like phenotypes in mice and its implications for treatment

Systemic Lupus Erythematosus (SLE) is a challenging autoimmune disease with limited treatment options. It arises from a combination of environmental factors and mutations in specific genes, including the Dnase1L3 gene. Dnase1L3, released by myeloid cells into the bloodstream, plays a crucial role in breaking down DNA within apoptotic bodies and chromatin, thereby reducing autoantibody formation and preventing SLE. However, there are several unknowns in the Dnase1L3 field, this project focuses on addressing key challenges in the study of Dnase1L3. Firstly, there is a need for specific activity assays that can differentiate between Dnase1L3 and Dnase1. Additionally, the importance of a minimum threshold concentration of Dnase1L3 activity in disease prevention remains unknown. Moreover, the impact of increasing the size of Dnase1L3 through PEGylation has not been explored in preclinical and serum half-life studies. To shed light on the role of partial Dnase1L3 loss in disease mechanisms, the project aims to utilize lupus murine models. The main objective is to develop functional assays for measuring Dnase1L3 specific activity. Furthermore, the project seeks to assess the effects of macrophage derived Dnase1L3 loss on specific cell types, such as macrophages, using conditional knockout mouse models. Finally, to evaluate Dnase1L3 as a potential therapeutic, the project aims to identify the lead candidate and determine its serum half-life. By addressing these objectives, this research aims to enhance our understanding of Dnase1L3 and its potential implications in disease prevention and treatment. In aim 1 I developed specific activity assays for Dnase1 and Dnase1L3. The plasmid digestion assay proved highly specific for Dnase1, while the immune complex degradation assay reliably measured Dnase1L3 activity. Next, to measure the impact of reduced Dnase1L3 protein levels were investigated through conditional knockout mice lacking Dnase1L3 in macrophages. These mice exhibited reduced serum Dnase1L3 levels, along with lupus-like symptoms such as increased autoantibodies and kidney pathology. The findings suggest that intermediate reduction in serum Dnase1L3 is sufficient to cause mild lupus phenotypes, highlighting Dnase1L3 as a key therapeutic target for lupus. Exploring strategies for enhancing Dnase1L3 enzyme replacement therapy, pegylated Dnase1L3 mutants have been evaluated and shown to have preserved activity. Serum half-life studies in lupus mouse models demonstrated prolonged stability of pegylated Dnase1L3, suggesting that PEGylation could improve the pharmacokinetics and reduce immunogenicity of Dnase1L3 enzyme replacement therapy in SLE. In conclusion, this study sheds light on the role of Dnase1L3 in SLE pathogenesis, the consequences of reduced Dnase1L3 activity, and strategies for enhancing Dnase1L3 enzyme replacement therapy. These findings contribute to a better understanding of the mechanisms underlying SLE and offer potential avenues for targeted treatment in autoimmune diseases

Spatial correlation function of intensity variations in the ground scintillation pattern produced by equatorial spread-F irregularities

23-32A theoretical model is used to relate the spatial variations found in intensity scintillation patterns formed on the ground due to scattering of VHF radio waves by equatorial ionospheric irregularities, with the spatial structure of these irregularities. As equatorial ionospheric irregularities are closely aligned with the geomagnetic field, they may be considered to constitute a two-dimensional dispersive random medium. Electron density variations, which produce refractive index irregularities in the medium, are assumed to be characterized by a power law spectrum. A numerical solution of- the equation satisfied by the fourth moment of intensity variations, in the plane of the receiver, is obtained using the split step method. The S4-index, which is the standard deviation of normalized intensity variations as well as spatial correlation function of intensity, is obtained by considering special cases of the fourth moment of intensity variations. Variation of S4-index with the standard deviation of phase fluctuations imposed by the ionospheric irregularities is studied for irregularities with different power spectral indices. Effect of varying phase fluctuations on the 50% de-correlation scale length is also studied for weak as well as strong scintillations. The S4-index and spatial scale lengths in the ground scintillation pattern depend on parameters like thickness and height of the irregularity layer, background plasma density, standard deviation of electron density fluctuation and irregularity power spectrum. The theoretical model is used to understand the roles of these parameters in determining the S4-index and spatial correlation function of intensity in the ground scintillation pattern

Spatial correlation function of intensity variations in the ground scintillation pattern produced by equatorial spread-F irregularities

A theoretical model is used to relate the spatial variations found in intensity scintillation patterns formed on the ground due to scattering of VHF radio waves by equatorial ionospheric irregularities, with the spatial structure of these irregularities. As equatorial ionospheric irregularities are closely aligned with the geomagnetic field, they may be considered to constitute a two-dimensional dispersive random medium. Electron density variations, which produce refractive index irregularities in the medium, are assumed to be characterized by a power law spectrum. A numerical solution of the equation satisfied by the fourth moment of intensity variations, in the plane of the receiver, is obtained using the split step method. The S4-index, which is the standard deviation of normalized intensity variations as well as spatial correlation function of intensity, is obtained by considering special cases of the fourth moment of intensity variations. Variation of S4-index with the standard deviation of phase fluctuations imposed by the ionospheric irregularities is studied for irregularities with different power spectral indices. Effect of varying phase fluctuations on the 50% de-correlation scale length is also studied for weak as well as strong scintillations. The S4-index and spatial scale lengths in the ground scintillation pattern depend on parameters like thickness and height of the irregularity layer, background plasma density, standard deviation of electron density fluctuation and irregularity power spectrum. The theoretical model is used to understand the roles of these parameters in determining the S4-index and spatial correlation function of intensity in the ground scintillation pattern

Deletion of Dnase1L3 from Macrophages results in reduced serum Dnase activity in mice

Abstract Dnase1L3 deficiency leads to 100% penetrance of pediatric onset Systemic lupus erythematous (SLE) while reduce Dnase1L3 levels is seen in adult-onset. Dnase1L3 is an endonuclease primarily secreted by myeloid cells like macrophages into the serum, where it degrades apoptotic bodies and chromatin, which reduces auto antibody formation and prevents SLE in both humans and mice. Importantly, reduction in the Dnase1L3 protein levels occurs both in adult-onset SLE in humans, and in polygenic murine models. This suggests that different levels of Dnase1L3 contribute to SLE progression. Also, decreasing Dnase1L3 levels leads to progressively worse SLE. However, the amount of serum Dnase1L3 needed, or whether the impact of Dnase1L3 is analog or digital remains unknown. In order to reduce Dnase1L3 protein levels, we generated Dnase1L3fl/fl x LysM-cre+/− conditional knockout (cKO) mice on B6 background. We determined the contribution of macrophage Dnase1L3 by measuring the Dnase1L3 protein levels and enzymatic activity by Dnase activity assay. We found that cKO mice showed ~2-log reduction in the serum Dnase activity. These cKO mice represent a cell-specific model to study the impact of Dnase1L3 protein levels on SLE pathogenesis. Together these findings suggest that macrophage specific Dnase1L3 may be a key player in lupus, however there could be other nucleases involved which we plan to inhibit using certain specific inhibitors. Overall, our findings suggest that macrophage specific Dnase1L3 is critical to prevent autoantibody formation in protecting lupus onset</jats:p

Solar flux dependence of coherence scales in scintillation patterns produced by ESF irregularities

The coherence scale length, defined as the 50% decorrelation scale length along the magnetic east-west direction, in the ground scintillation pattern obtained at a dip equatorial location, due to scattering of VHF radio waves by equatorial spread F (ESF) irregularities, is calculated, using amplitude scintillation data recorded by two spaced receivers. The average east-west drift of the ground scintillation pattern, during the pre- and post-midnight periods, also calculated from the same observations, shows an almost linear increase with 10.7-cm solar flux. In the present paper the variability of the drift is automatically taken into account in the calculation of the coherence scale length of the ground scintillation pattern. For weak scintillations, the coherence scale depends on the Fresnel scale, which varies with the height of the irregularity layer, and also on the spectral index of the irregularity power spectrum. It is found that for weak scintillations, the coherence scales are much better organized according to the 10.7-cm solar flux, during the pre-midnight period, than during the post-midnight period, with a general trend of coherence scale length increasing with 10.7-cm solar flux except for cases with F 10.7-cm solar flux &lt;100. This indicates that, during the initial phase of ESF irregularity development, the irregularity spectrum does not have much variability while further evolution of the spatial structure in ESF irregularities is controlled by factors other than the solar flux

Dnase1L3 levels from macrophages in providing protection against SLE development

Abstract A hallmark symptom of SLE is elevated autoantibodies, especially anti-dsDNA antibodies in multiple organs and tissues. Deficiency of Dnase1L3, a serum endonuclease, allows the accumulation of self-DNA leading to autoantibody generation. The source of Dnase1L3 is primarily expressed by macrophages and dendritic cells. Complete Dnase1L3 deficiency is known to promote the SLE phenotype, however, the dosage required to prevent disease onset is unknown. We hypothesize that macrophages provide sufficient Dnase1L3 expression to regulate autoantibody levels in the host’s system. We generated conditional, macrophage specific Dnase1L3 knockout mice to assess disease progression. We collected serum samples from 6–50 weeks of age and measured total IgG and anti-dsDNA levels by ELISA to develop a longitudinal analysis of autoantibody elevation. At sacrifice, we observed pathology and measured IgG deposition in the kidney and heart by immunofluorescence. We observed that the elimination of Dnase1L3 in macrophages increased anti-dsDNA antibodies in the conditional knockout mice. In addition, we observed minor abnormalities in the glomeruli size for the conditional knockout mice as compared to the wild type mice. Our results indicate that macrophages are capable of providing protection against the development of SLE.</jats:p