Preclinical Evidence for the Glucocorticoid-Sparing Potential of a Dual Toll-Like Receptor 7/8 Inhibitor in Autoimmune Diseases.

Toll-like receptor 7 (TLR7) and TLR8 are single-stranded RNA-sensing endosomal pattern recognition receptors that evolved to defend against viral infections. However, aberrant TLR7/8 activation by endogenous ligands has been implicated in the pathogenesis of autoimmune diseases including systemic lupus erythematosus. TLR activation and type I interferon (IFN) were shown recently to impart resistance to glucocorticoids (GC), which are part of the standard of care for multiple autoimmune diseases. While GCs are effective, a plethora of undesirable effects limit their use. New treatment approaches that allow for the use of lower and safer doses of GCs would be highly beneficial. Herein, we report that a dual TLR7/8 inhibitor (TLR7/8i) increases the effectiveness of GCs in inflammatory settings. Human peripheral blood mononuclear cell studies revealed increased GC sensitivity in the presence of TLR7/8i for reducing inflammatory cytokine production, a synergistic effect that was most pronounced in myeloid cells, particularly monocytes. Gene expression analysis by NanoString and single-cell RNA sequencing revealed that myeloid cells were substantially impacted by combining low-dose TLR7/8i and GC, as evidenced by the effects on nuclear factor-kappa B-regulated cytokines and GC-response genes, although IFNs were affected to a smaller degree. Low dose of TLR7/8i plus GC was more efficacious then either agent alone in the MRL/lpr mouse model of lupus, with improved proteinuria and survival. Overall, our findings indicate a GC-sparing potential for TLR7/8i compounds, suggesting TLR7/8i may offer a new strategy for the treatment of autoimmune diseases. SIGNIFICANCE STATEMENT: Some features of autoimmune diseases may be resistant to glucocorticoids, mediated at least in part by toll-like receptor (TLR) activation, necessitating higher doses that are associated with considerable toxicities. We demonstrate that TLR7/8 inhibition and glucocorticoids work synergistically to reduce inflammation in a cell-type specific manner and suppress disease in a mouse model of lupus. TLR7/8 inhibition is a promising strategy for the treatment of autoimmune diseases and has glucocorticoid-sparing potential.

Genomic Identification of Low-Density Granulocytes and Analysis of Their Role in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of low-density granulocytes (LDGs) with a heightened capacity for spontaneous NETosis, but the contribution of LDGs to SLE pathogenesis remains unclear. To characterize LDGs in human SLE, gene expression profiles derived from isolated LDGs were characterized by weighted gene coexpression network analysis, and a 92-gene module was identified. The LDG gene signature was enriched in genes related to neutrophil degranulation and cell cycle regulation. This signature was assessed in gene expression datasets from two large-scale SLE clinical trials to study associations between LDG enrichment, SLE manifestations, and treatment regimens. LDG enrichment in the blood was associated with corticosteroid treatment as well as anti-dsDNA, low serum complement, renal manifestations, and vasculitis, but the latter two of these associations were dependent on concomitant corticosteroid treatment. In addition, LDG enrichment was associated with enrichment of gene signatures induced by type I IFN and TNF irrespective of corticosteroid treatment. Notably, LDG enrichment was not found in numerous tissues affected by SLE. Comparison with relevant reference datasets indicated that LDG enrichment is likely reflective of increased granulopoiesis in the bone marrow and not peripheral neutrophil activation. The results have uncovered important determinants of the appearance of LDGs in SLE and have emphasized the likely role of LDGs in specific aspects of lupus pathogenesis.

Human dendritic cells exhibit a pronounced type I IFN signature following Leishmania major infection that is required for IL-12 induction.

Leishmania major-infected human dendritic cells (DCs) exhibit a marked induction of IL-12, ultimately promoting a robust Th1-mediated response associated with parasite killing and protective immunity. The host cell transcription machinery associated with the specific IL-12 induction observed during L. major infection remains to be thoroughly elucidated. In this study, we used Affymetrix GeneChip (Affymetrix) to globally assess the host cell genes and pathways associated with early L. major infection in human myeloid-derived DCs. Our data revealed 728 genes were significantly differentially expressed and molecular signaling pathway revealed that the type I IFN pathway was significantly enriched. Addition of a neutralizing type I IFN decoy receptor blocked the expression of IRF7 and IL-12p40 during DC infection, indicating the L. major-induced expression of IL-12p40 is dependent upon the type I IFN signaling pathway. In stark contrast, IL-12p40 expression is not elicited by L. donovani, the etiological agent of deadly visceral leishmaniasis. Therefore, we examined the gene expression profile for several IFN response genes in L. major versus L. donovani DC infections. Our data revealed that L. major, but not L. donovani, induces expression of IRF2, IRF7, and IFIT5, implicating the regulation of type I IFN-associated signaling pathways as mediating factors toward the production of IL-12.

The position of repetitive DNA sequence in the southern cattle tick genome permits chromosome identification.

Fluorescent in-situ hybridization (FISH) using meiotic chromosome preparations and highly repetitive DNA from the southern cattle tick, Rhipicephalus microplus, was undertaken to investigate genome organization. Several classes of highly repetitive DNA elements were identified by screening a R. microplus bacterial artificial chromosome (BAC) library. A repeat unit of approximately 149 bp, RMR-1 was localized to the subtelomeric regions of R. microplus autosomes 1-6 and 8-10. A second repeat unit, RMR-2 was localized to the subtelomeric regions of all autosomes and the X chromosome. RMR-2 was composed of three distinct repeat populations, RMR-2a, RMR-2b and RMR-2c of 178, 177 and 216 bp in length, respectively. Localization of an rDNA probe identified a single nucleolar organizing region on one autosome. Using a combination of labeled probes, we developed a preliminary karyotype for R. microplus. We present evidence that R. microplus has holocentric chromosomes and explore the implications of these findings for tick chromosome biology and genomic research.

Analysis of gene expression from systemic lupus erythematosus synovium reveals myeloid cell-driven pathogenesis of lupus arthritis.

Arthritis is a common manifestation of systemic lupus erythematosus (SLE) yet understanding of the underlying pathogenic mechanisms remains incomplete. We, therefore, interrogated gene expression profiles of SLE synovium to gain insight into the nature of lupus arthritis (LA), using osteoarthritis (OA) and rheumatoid arthritis (RA) as comparators. Knee synovia from SLE, OA, and RA patients were analyzed for differentially expressed genes (DEGs) and also by Weighted Gene Co-expression Network Analysis (WGCNA) to identify modules of highly co-expressed genes. Genes upregulated and/or co-expressed in LA revealed numerous immune/inflammatory cells dominated by a myeloid phenotype, in which pathogenic macrophages, myeloid-lineage cells, and their secreted products perpetuate inflammation, whereas OA was characterized by fibroblasts and RA of lymphocytes. Genes governing trafficking of immune cells into the synovium by chemokines were identified, but not in situ generation of germinal centers (GCs). Gene Set Variation Analysis (GSVA) confirmed activation of specific immune cell types in LA. Numerous therapies were predicted to target LA, including TNF, NFκB, MAPK, and CDK inhibitors. Detailed gene expression analysis identified a unique pattern of cellular components and physiologic pathways operative in LA, as well as drugs potentially able to target this common manifestation of SLE.

Patient ancestry significantly contributes to molecular heterogeneity of systemic lupus erythematosus.

Gene expression signatures can stratify patients with heterogeneous diseases, such as systemic lupus erythematosus (SLE), yet understanding the contributions of ancestral background to this heterogeneity is not well understood. We hypothesized that ancestry would significantly influence gene expression signatures and measured 34 gene modules in 1566 SLE patients of African ancestry (AA), European ancestry (EA), or Native American ancestry (NAA). Healthy subject ancestry-specific gene expression provided the transcriptomic background upon which the SLE patient signatures were built. Although standard therapy affected every gene signature and significantly increased myeloid cell signatures, logistic regression analysis determined that ancestral background significantly changed 23 of 34 gene signatures. Additionally, the strongest association to gene expression changes was found with autoantibodies, and this also had etiology in ancestry: the AA predisposition to have both RNP and dsDNA autoantibodies compared with EA predisposition to have only anti-dsDNA. A machine learning approach was used to determine a gene signature characteristic to distinguish AA SLE and was most influenced by genes characteristic of the perturbed B cell axis in AA SLE patients.

Gene expression analysis delineates the potential roles of multiple interferons in systemic lupus erythematosus.

A role for interferon (IFN) in systemic lupus erythematosus (SLE) pathogenesis is inferred from the prominent IFN gene signature (IGS), but the major IFN species and its relationship to disease activity are unknown. A bioinformatic approach employing individual IFN species gene signatures to interrogate SLE microarray datasets demonstrates a putative role for numerous IFN species, with prominent expression of IFNB1 and IFNW signatures. In contrast with other SLE-affected organs, the IGS is less prominent in lupus nephritis. SLE patients with active and inactive disease have readily detectable IGS and the IGS changes synchronously with a monocyte signature but not disease activity, and is significantly related to monocyte transcripts. Monocyte over-expression of three times as many IGS transcripts as T and B cells and IGS retention in monocytes, but not T and B cells from inactive SLE patients contribute to the lack of correlation between the IGS and SLE disease activity.

Machine learning approaches to predict lupus disease activity from gene expression data.

The integration of gene expression data to predict systemic lupus erythematosus (SLE) disease activity is a significant challenge because of the high degree of heterogeneity among patients and study cohorts, especially those collected on different microarray platforms. Here we deployed machine learning approaches to integrate gene expression data from three SLE data sets and used it to classify patients as having active or inactive disease as characterized by standard clinical composite outcome measures. Both raw whole blood gene expression data and informative gene modules generated by Weighted Gene Co-expression Network Analysis from purified leukocyte populations were employed with various classification algorithms. Classifiers were evaluated by 10-fold cross-validation across three combined data sets or by training and testing in independent data sets, the latter of which amplified the effects of technical variation. A random forest classifier achieved a peak classification accuracy of 83 percent under 10-fold cross-validation, but its performance could be severely affected by technical variation among data sets. The use of gene modules rather than raw gene expression was more robust, achieving classification accuracies of approximately 70 percent regardless of how the training and testing sets were formed. Fine-tuning the algorithms and parameter sets may generate sufficient accuracy to be informative as a standalone estimate of disease activity.

Duration of chronic inflammation alters gene expression in muscle from untreated girls with juvenile dermatomyositis.

BACKGROUND: To evaluate the impact of the duration of chronic inflammation on gene expression in skeletal muscle biopsies (MBx) from untreated children with juvenile dermatomyositis (JDM) and identify genes and biological processes associated with the disease progression, expression profiling data from 16 girls with active symptoms of JDM greater than or equal to 2 months were compared with 3 girls with active symptoms less than 2 months. RESULTS: Seventy-nine genes were differentially expressed between the groups with long or short duration of untreated disease. Genes involved in immune responses and vasculature remodelling were expressed at a higher level in muscle biopsies from children with greater or equal to 2 months of symptoms, while genes involved in stress responses and protein turnover were expressed at a lower level. Among the 79 genes, expression of 9 genes showed a significant linear regression relationship with the duration of untreated disease. Five differentially expressed genes--HLA-DQA1, smooth muscle myosin heavy chain, clusterin, plexin D1 and tenomodulin--were verified by quantitative RT-PCR. The chronic inflammation of longer disease duration was also associated with increased DC-LAMP+ and BDCA2+ mature dendritic cells, identified by immunohistochemistry. CONCLUSION: We conclude that chronic inflammation alters the gene expression patterns in muscle of untreated children with JDM. Symptoms lasting greater or equal to 2 months were associated with dendritic cell maturation and anti-angiogenic vascular remodelling, directly contributing to disease pathophysiology.

Identification of alterations in macrophage activation associated with disease activity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by abnormalities in B cell and T cell function, but the role of disturbances in the activation status of macrophages (Mϕ) has not been well described in human patients. To address this, gene expression profiles from isolated lymphoid and myeloid populations were analyzed to identify differentially expressed (DE) genes between healthy controls and patients with either inactive or active SLE. While hundreds of DE genes were identified in B and T cells of active SLE patients, there were no DE genes found in B or T cells from patients with inactive SLE compared to healthy controls. In contrast, large numbers of DE genes were found in myeloid cells (MC) from both active and inactive SLE patients. Among the DE genes were several known to play roles in Mϕ activation and polarization, including the M1 genes STAT1 and SOCS3 and the M2 genes STAT3, STAT6, and CD163. M1-associated genes were far more frequent in data sets from active versus inactive SLE patients. To characterize the relationship between Mϕ activation and disease activity in greater detail, weighted gene co-expression network analysis (WGCNA) was used to identify modules of genes associated with clinical activity in SLE patients. Among these were disease activity-correlated modules containing activation signatures of predominantly M1-associated genes. No disease activity-correlated modules were enriched in M2-associated genes. Pathway and upstream regulator analysis of DE genes from both active and inactive SLE MC were cross-referenced with high-scoring hits from the drug discovery Library of Integrated Network-based Cellular Signatures (LINCS) to identify new strategies to treat both stages of SLE. A machine learning approach employing MC gene modules and a generalized linear model was able to predict the disease activity status in unrelated gene expression data sets. In summary, altered MC gene expression is characteristic of both active and inactive SLE. However, disease activity is associated with an alteration in the activation of MC, with a bias toward the M1 proinflammatory phenotype. These data suggest that while hyperactivity of B cells and T cells is associated with active SLE, MC potentially direct flare-ups and remission by altering their activation status toward the M1 state.

Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia.

Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beMφs) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beMφs to populate the niche and that the presence of beMφs does not alter behavior. Furthermore, beMφs maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beMφs as a unique CNS cell type and demonstrates that therapeutic engraftment of beMφs may be possible with irradiation-free conditioning regimens.

Variation in genome size of argasid and ixodid ticks.

The suborder Ixodida includes many tick species of medical and veterinary importance, but little is known about the genomic characteristics of these ticks. We report the first study to determine genome size in two species of Argasidae (soft ticks) and seven species of Ixodidae (hard ticks) using flow cytometry analysis of fluorescent stained nuclei. Our results indicate a large haploid genome size (1C>1000 Mbp) for all Ixodida with a mean of 1281 Mbp (1.31+/-0.07 pg) for the Argasidae and 2671 Mbp (2.73+/-0.04 pg) for the Ixodidae. The haploid genome size of Ixodes scapularis was determined to be 2262 Mbp. We observed inter- and intra-familial variation in genome size as well as variation between strains of the same species. We explore the implications of these results for tick genome evolution and tick genomics research.

Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells.

BACKGROUND: Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. METHODOLOGY/PRINCIPAL FINDINGS: Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. CONCLUSIONS/SIGNIFICANCE: These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

Profiling of human acquired immunity against the salivary proteins of Phlebotomus papatasi reveals clusters of differential immunoreactivity.

Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents.