The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (∼70 kb) plus a ∼5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps- 2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed open reading frames than the remainder of the genome. The mitochondrial plasmid shares a 251-bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that can affect multiple end organs. Kidney and brain are two of the organs most commonly involved in SLE. Past studies have suggested the importance of macrophages in the pathogenesis of lupus nephritis (LN). Furthermore, as the immune effectors of the brain, microglia have been implicated in pathways leading to neuropsychiatric SLE (NPSLE). We depleted macrophages and microglia using GW2580, a small colony stimulating factor-1 receptor (CSF-1R) kinase inhibitor, in MRL-lpr/lpr (MRL/lpr) mice, a classic murine lupus model that displays features of both LN and NPSLE. Treatment was initiated before the onset of disease, and mice were followed for the development of LN and neurobehavioral dysfunction throughout the study. Treatment with GW2580 significantly ameliorated kidney disease, as evidenced by decreased proteinuria, BUN, and improved renal histopathology, despite equivalent levels of IgG and C3 deposition in the kidneys of treated and control mice. We were able to confirm macrophage depletion within the kidney via IBA-1 staining. Furthermore, we observed specific improvement in the depression-like behavioral deficit of MRL/lpr mice with GW2580 treatment. Circulating antibody and autoantibody levels were, however, not affected. These results provide additional support for the role of macrophages as a potentially valuable therapeutic target in SLE. Inhibiting CSF-1 receptor signaling would be more targeted than current immunosuppressive therapies, and may hold promise for the treatment of renal and neuropsychiatric end organ disease manifestations.

Systemic lupus erythematosus (SLE) is a chronic, multiorgan, systemic autoimmune disease that is more common in women than men and is typically diagnosed during reproductive age, necessitating sex-specific considerations in care. In women there is no substantive evidence to suggest that SLE reduces fertility, but subfertility may occur as a result of active disease, immunosuppressive drugs, and age-related declines in fertility related to delays in childbearing. Although pregnancy outcomes have improved, SLE still poses risks in pregnancy that contribute to poorer maternal and fetal outcomes. Cyclophosphamide, an important agent for the treatment of severe or life-threatening lupus, may adversely affect fertility, particularly with increases in dose and patient age. Fertility

Immune-mediated glomerulonephritis is a serious end organ pathology that affects patients with systemic lupus erythematosus (SLE). A common murine model used to study lupus nephritis (LN) is nephrotoxic serum nephritis (NTN) in which mice are passively transferred nephrotoxic antibodies. We have previously shown that macrophages are important for the pathogenesis of LN. To further investigate the mechanism and determine if the contribution of macrophages is mediated via the NF-κB pathway we created B6 mice which had RelA knocked out in myeloid cells, thus inhibiting classical NF-κB signaling in this cell lineage. We induced NTN in this strain to assess the importance of macrophage derived NF-κB signaling in contributing to disease progression. Myeloid cell RelA knock out (KO) mice injected with nephrotoxic serum had significantly ameliorated proteinuria (p<0.01) and serum BUN levels (p<0.05) compared to control injected wildtype B6 mice (WT). Inhibiting myeloid NF-κB signaling also decreased inflammatory modulators within the kidneys. We measured significant decreases of IL-1a, IFNg, and IL-6 in kidneys from KO mice, but higher IL-10 expression. Flow cytometry revealed decreased numbers of kidney infiltrating classically activated macrophages in KO mice as well. Analysis of renal histopathology is pending. Taken together, our studies indicate that macrophage NF-κB signaling is instrumental in the contribution of this cell type to the pathogenesis of NTN. Our results suggest that while approaches which decrease macrophage numbers can be effective in immune mediated nephritis, more targeted treatments directed at modulating macrophage signaling and/or function could be beneficial, at least in the early stages of disease.

pathogenesis. The CXorf21 protein product, known as TASL, binds SLC15a4, another gene with SLE-risk alleles, on the surface of the endolysosome. Methods We used CRIPR-Cas to knock down expression of CXorf21 (TASL) in primary monocytes, B cells and T cells. We studied lysomomal pH using LysoSensor Yellow/Blue DND-160 and pHrodo Red. We measured cytokines by ELISA of cell culture supernats. We used an assay for NADP/NADPH conversion to determine whether TASL is a short chain dehydrogenase. Finally, we typed SLE subjects and controls for the lupus-associated single nucleotide polymorphism within CXorf21.Results Knock out of Slc15a4 abrogates TLR7 signaling. Our data demonstrate that CRISPR-cas knock down of TASL expression renders lysosomal pH more alkalotic, which should impair TLR7 signaling.Furthermore, in immune cells expressing TASL (monocytes and B lymphocytes), lysosomal pH was lower in female cells compared to male cells. In female B lymphocytes or monocytes, CRISPR-cas knock down of TASL expression markedly reduced cytokine secretion in response to TLR7 ligand engagement. The TASL protein has several features suggesting it is a short chain dehydrogenase. Our functional studies indicate that TASL catalyzes conversion of NADP to NADPH. Genetic studies demonstrate synergistic interaction among CXorf21 and Slc15a4 for SLE risk. Conclusions The CXorf21 gene is differentially expressed in female and male immune cells based on escape from X inactivation and is critical for lysosomal pH and TLR7 signaling. Risk of SLE may be enhanced among individuals with risk alleles at both CXorf21 and Slc15a4. Sex bias in SLE is attributable to an X chromosome dose effect, which may be mediated in part by CXorf21 and its protein product TASL.