Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis.

OBJECTIVES: Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of SLE. Despite evidence showing the importance of platelet-neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and Toll-like receptor 7 (TLR7) in clinical disease. METHODS: Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7 mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet-HDN mixing studies using TLR7-deficient mice and Klinefelter syndrome patients. RESULTS: SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the peripheral blood mononuclear cell (PBMC) layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7 and that the association results in increased NETosis. The neutrophil:platelet ratio is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of LN. CONCLUSIONS: LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils that may be an important therapeutic opportunity for LN.

Assessing Lupus-Like Disease in Murine Model Systems.

Systemic Lupus Erythematosus (SLE) is a complex and heterogenous autoimmune disease, where genetics, immunology, and environmental factors all play a role. Murine models have contributed critical information on mechanisms of disease and prospective therapeutics. The key features that have been used to study the disease include the development of anti-nuclear autoantibodies (ANAs), splenomegaly, and kidney disease. The loss of tolerance and subsequent autoimmune features, and the progression to severe disease, are all dependent on immune dysregulation. In this article, we will describe the methods used to evaluate the underlying immunological features of the disease, as a more sensitive strategy to understand the disease itself and the mechanisms of potential novel therapeutics. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: End study protocols for tissue harvesting Basic Protocol 2: End study protocols for tissue processing Basic Protocol 3: Immunophenotyping using flow cytometry protocols Support Protocol: Tissue processing for cold storage Basic Protocol 4: Additional tissue processing for later analyses Basic Protocol 5: Analysis of serum auto-antibodies by ELISAs (ANAs, snRNP, and dsDNA).

CD34+CD133+CD309+ circulating angiogenic cell level is reduced but positively related to hydroxychloroquine use in SLE patients-a case-control study and meta-regression analysis.

OBJECTIVES: To identify and quantify the level of CD34+CD133+CD309+ circulating angiogenic cells (CAC) and explore factors associated with the level of CAC in patients with SLE. METHODS: The peripheral blood mononuclear cells of consecutive SLE patients and demographically matched healthy controls (HC) were extracted and identified, enumerated and compared for CAC levels by multi-colour flow cytometry based on the EULAR Scleroderma Trials and Research (EUSTAR) recommendation. Meta-analyses were performed by combining the current and previous case-control studies, aiming to increase the statistical power to discern the difference in CAC level between SLE patients and HC. Mixed-model meta-regression was conducted to explore potential demographic and clinical factors that were associated with CAC level. RESULTS: A lower level of CAC was found in 29 SLE patients compared with 24 HC [mean (s.d.) 10.76 (13.9) vs 24.58 (25.4) cells/ml, P = 0.015]. Random-effects meta-analyses of the current and six previously published case-control studies involving 401 SLE patients and 228 HC revealed a lower CAC level compared with HC (standardized mean difference = -2.439, P = 0.001). Meta-regression analysis demonstrated that HCQ use was associated with a more discrepant CAC level between both groups (P = 0.01115). CONCLUSION: SLE patients had a significantly lower CD34+CD133+CD309+ CAC level than HC, and HCQ use was associated with a more discrepant CAC level between SLE patients and HC. This study triggers further observational, interventional and mechanistic studies to address the beneficial impact of HCQ on the functionality of CAC in SLE patients.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

TLR7 Protein Expression in Mild and Severe Lupus-Prone Models Is Regulated in a Leukocyte, Genetic, and IRAK4 Dependent Manner.

The global increase in autoimmunity, together with the emerging autoimmune-related side effects of cancer immunotherapy, have furthered a need for understanding of immune tolerance and activation. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disease, affecting multiple organs, and tissues. Studying SLE creates knowledge relevant not just for autoimmunity, but the immune system in general. Murine models and patient studies have provided increasing evidence for the innate immune toll like receptor-7 (TLR7) in disease initiation and progression. Here, we demonstrated that the kinase activity of the TLR7-downstream signaling molecule, interleukin-1 receptor associated kinase 4 (IRAK4), is essential for mild and severe autoimmune traits of the Sle1 and Sle1-TLR7 transgenic (Sle1Tg7) murine models, respectively. Elimination of IRAK4 signaling prevented all pathological traits associated with murine lupus, including splenomegaly with leukocyte expansion, detectable circulating antinuclear antibodies and glomerulonephritis, in both Sle1 and Sle1Tg7 mice. The expansion of germinal center B cells and increased effector memory T cell phenotypes that are typical of lupus-prone strains, were also prevented with IRAK4 kinase elimination. Analysis of renal leukocyte infiltrates confirmed our earlier findings of an expanded conventional dendritic cell (cDC) within the kidneys of nephritic mice, and this was prevented with IRAK4 kinase elimination. Analysis of TLR7 at the protein level revealed that the expression in immune cells is dependent on the TLR7-transgene itself and/or autoimmune disease factors in a cell-specific manner. Increased TLR7 protein expression in renal macrophages and cDCs correlated with disease parameters such as blood urea nitrogen (BUN) levels and the frequency of leukocytes infiltrating the kidney. These findings suggest that controlling the level of TLR7 or downstream signaling within myeloid populations may prevent chronic inflammation and severe nephritis.

A Flow Cytometry-Based Assay for High-Throughput Detection and Quantification of Neutrophil Extracellular Traps in Mixed Cell Populations.

Neutrophil extracellular traps (NETs) are web-like structures composed of decondensed chromatin and antimicrobial proteins that are released into the extracellular space during microbial infections. This active cell death program is known as NETosis. To date, florescence microscopy is the widely accepted method for visualization and quantification of NETs. However, this method is subjective, time consuming and yields low numbers of analyzed polymorphonuclear cells (PMNs) per sample. Increasing interest has emerged on the identification of NETs using flow cytometry techniques. However, flow cytometry analysis of NETs requires particular precautions for sample preparation to obtain reproducible data. Herein, we describe a flow cytometry-based assay for high-throughput detection and quantification of NETosis in mixed cell populations. We used fluorescent-labeled antibodies against cell markers on PMNs together with a combination of nucleic acid stains to measure NETosis in whole blood (WB) and purified PMNs. Using plasma membrane-impermeable DNA-binding dye, SYTOX Orange (SO), we found that cell-appendant DNA of NETting PMNs were positive for SO and DAPI. The combination of optimally diluted antibody and nucleic acid dyes required no washing and yielded low background fluorescence. Significant correlations were found for NETosis from WB and purified PMNs. We then validated the assay by comparing with time-lapse live cell fluorescence microscopy and determined very good intraassay and interassay variances. The assay was then applied to a disease associated with NETosis, systemic lupus erythematosus (SLE). We examined PMA-induced NETosis in peripheral PMNs from SLE patients and controls and in bone marrow PMNs from multiple murine models. In summary, this assay is observer-independent and allows for rapid assessment of a large number of PMNs per sample. Use of this assay does not require sophisticated microscopic equipment like imaging flow cytometers and may be a starting point to analyze extracellular trap formation from immune cells other than PMNs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Toll-Like Receptor 9 Deficiency Breaks Tolerance to RNA-Associated Antigens and Up-Regulates Toll-Like Receptor 7 Protein in Sle1 Mice.

OBJECTIVE: Toll-like receptors (TLRs) 7 and 9 are important innate signaling molecules with opposing roles in the development and progression of systemic lupus erythematosus (SLE). While multiple studies support the notion of a dependency on TLR-7 for disease development, genetic ablation of TLR-9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. This study was undertaken to examine the suppressive role of TLR-9 in the development of severe lupus in a mouse model. METHODS: We crossed Sle1 lupus-prone mice with TLR-9-deficient mice to generate Sle1TLR-9-/- mice. Mice ages 4.5-6.5 months were evaluated for severe autoimmunity by assessing splenomegaly, GN, immune cell populations, autoantibody and total Ig profiles, kidney dendritic cell (DC) function, and TLR-7 protein expression. Mice ages 8-10 weeks were used for functional B cell studies, Ig profiling, and determination of TLR-7 expression. RESULTS: Sle1TLR-9-/- mice developed severe disease similar to TLR-9-deficient MRL and Nba2 models. Sle1TLR-9-/- mouse B cells produced more class-switched antibodies, and the autoantibody repertoire was skewed toward RNA-containing antigens. GN in these mice was associated with DC infiltration, and purified Sle1TLR-9-/- mouse renal DCs were more efficient at TLR-7-dependent antigen presentation and expressed higher levels of TLR-7 protein. Importantly, this increase in TLR-7 expression occurred prior to disease development, indicating a role in the initiation stages of tissue destruction. CONCLUSION: The increase in TLR-7-reactive immune complexes, and the concomitant enhanced expression of their receptor, promotes inflammation and disease in Sle1TLR9-/- mice.

Brief report: Decreased expression of CD244 (SLAMF4) on monocytes and platelets in patients with systemic lupus erythematosus.

The signalling lymphocyte activation molecule (SLAM) family receptors play important roles in modulating immune responses. Previous studies in murine models and patients have suggested an association of the SLAM family (SLAMF) members with the development of autoimmunity, particularly systemic lupus erythematosus (SLE). Since previous investigations on CD244 expression have focussed on NK and T cells, the aim of this study was to evaluate the surface expression of major SLAMF members across monocytes and polymorphonuclear cells in an Asian SLE cohort and explore their potential associations with SLE-related disease activity and autoantibodies. Thirty-nine SLE patients and twenty-nine healthy controls (HC) were evaluated for the expression of CD150, CD84, CD229, CD48, CD244, CD352 and CD319. We determined a significantly lower expression of CD244 on monocytes in SLE patients compared to HC. Furthermore, monocyte CD244 expression was negatively associated with several serum autoantibody titres. Our findings suggest that this molecule plays an important role in immune tolerance mechanisms and should be investigated further.

Monocyte Siglec-14 expression is upregulated in patients with systemic lupus erythematosus and correlates with lupus disease activity.

Objective: Siglecs are sialic acid-binding immunoglobulin-like lectins expressed on the surface of immune cells, which participate in the discrimination of self and non-self. We investigated myeloid CD33-related Siglec expression in a cohort of patients with SLE. Methods: Cell surface expression of Siglec-5/14, Siglec-9 and Siglec-10 on peripheral myeloid subsets were analysed from 39 SLE patients using flow cytometry. Genotyping of the Siglec-5/14 locus was also performed. Clinical markers of SLE disease activity, including SLEDAI, serum complement concentrations and serum autoantibodies, were assessed and correlated with Siglec levels. Results: Siglec-14 expression on SLE monocytes (median = 518, interquartile range: 411) was significantly higher when compared with healthy controls (median = 427, interquartile range: 289.3; P < 0.05) and correlated positively with SLEDAI scoring and anti-Sm and anti-SSB autoantibodies (P < 0.05). A negative correlation was determined with patient serum C3 concentrations (P < 0.005). Genotyping of the Siglec-5/14 locus revealed a high frequency of the Siglec-14 null allele across both groups, reflecting the incidence in Asian populations. Conclusion: Our data suggest that the Siglec immunomodulatory molecules, in particular Siglec-14 expression on monocytes, may play an important role in the inflammatory events of SLE. No bias was found with regard to SIGLEC14 genotype in our patient group compared with healthy controls. Larger comparisons of mixed ethnicity might, however, reveal an important role for Siglecs in the pathogenesis of autoimmune disease.

TLR7 and TLR9 ligands regulate antigen presentation by macrophages.

The toll-like receptors (TLRs) are important innate receptors recognizing potentially pathogenic material. However, they also play a significant role in the development of Alzheimer's disease, cancer, autoimmunity and the susceptibility to viral infections. Macrophages are essential for an effective immune response to foreign material and the resolution of inflammation. In these studies, we examined the impact of different TLR ligands on macrophage cell function. We demonstrate that stimulation of all TLRs tested increases the phagocytosis of apoptotic cells by macrophages. TLR7 and TLR9 ligation decreased the levels of the surface co-expression molecules CD86 and MHCII, which was associated with a concomitant reduction in antigen presentation and proliferation of T cells. This down-regulation in macrophage function was not due to an increase in cell death. In fact, exposure to TLR7 or TLR9 ligands promoted cell viability for up to 9 days, in contrast to TLR3 or TLR4. Additionally, macrophages exposed to TLR7/TLR9 ligands had a significantly lower ratio of Il-12/Il-10 mRNA expression compared with those treated with the TLR4 ligand, LPS. Taken together, these data demonstrate that TLR7/TLR9 ligands push the macrophage into a phagocytic long-lived cell, with a decreased capacity of antigen presentation and reminiscent of the M2 polarized state.

RNA sensing by conventional dendritic cells is central to the development of lupus nephritis.

Glomerulonephritis is a common and debilitating feature of systemic lupus erythematosus (SLE). The precise immune mechanisms that drive the progression from benign autoimmunity to glomerulonephritis are largely unknown. Previous investigations have shown that a moderate increase of the innate Toll-like receptor 7 (TLR7) is sufficient for the development of nephritis. In these systems normalization of B-cell TLR7 expression or temporal depletion of plasmacytoid dendritic cells (pDCs) slow progression; however, the critical cell that is responsible for driving full immunopathology remains unidentified. In this investigation we have shown that conventional DC expression of TLR7 is essential for severe autoimmunity in the Sle1Tg7 model of SLE. We show that a novel expanding CD11b(+) conventional DC subpopulation dominates the infiltrating renal inflammatory milieu, localizing to the glomeruli. Moreover, exposure of human myeloid DCs to IFN-α or Flu increases TLR7 expression, suggesting they may have a role in self-RNA recognition pathways in clinical disease. To our knowledge, this study is the first to highlight the importance of conventional DC-TLR7 expression for kidney pathogenesis in a murine model of SLE.

Enhanced neutralizing antibody titers and Th1 polarization from a novel Escherichia coli derived pandemic influenza vaccine.

Influenza pandemics can spread quickly and cost millions of lives; the 2009 H1N1 pandemic highlighted the shortfall in the current vaccine strategy and the need for an improved global response in terms of shortening the time required to manufacture the vaccine and increasing production capacity. Here we describe the pre-clinical assessment of a novel 2009 H1N1 pandemic influenza vaccine based on the E. coli-produced HA globular head domain covalently linked to virus-like particles derived from the bacteriophage Qß. When formulated with alum adjuvant and used to immunize mice, dose finding studies found that a 10 µg dose of this vaccine (3.7 µg globular HA content) induced antibody titers comparable to a 1.5 µg dose (0.7 µg globular HA content) of the licensed 2009 H1N1 pandemic vaccine Panvax, and significantly reduced viral titers in the lung following challenge with 2009 H1N1 pandemic influenza A/California/07/2009 virus. While Panvax failed to induce marked T cell responses, the novel vaccine stimulated substantial antigen-specific interferon-γ production in splenocytes from immunized mice, alongside enhanced IgG2a antibody production. In ferrets the vaccine elicited neutralizing antibodies, and following challenge with influenza A/California/07/2009 virus reduced morbidity and lowered viral titers in nasal lavages.

Ductal carcinoma in situ of the breast in Singapore: recent trends and clinical implications.

BACKGROUND: Breast ductal carcinoma in-situ (DCIS) is increasingly being diagnosed as a result of screening mammography and better pathological recognition. With this and the rising breast cancer incidence in Singapore, DCIS is poised to become a bigger part of surgical practice. Principles of screening, diagnosis and management of DCIS have also been rapidly evolving. Against this background, a clinicopathological audit of recent cases of DCIS in our centre was performed. METHODS: Thirty-eight cases of DCIS diagnosed in the period 1997-2000 were retrospectively analysed. Histological examination and immunohistochemical studies for oestrogen and progesterone receptor expression were performed. RESULTS: In the present study, DCIS was most common in the group of patients who were <50 years (58%) as compared to an American series' where the corresponding group were > or =60 years (36%). Compared to a previous local study a decade ago, the present series showed that: (i) DCIS constituted a higher proportion of all breast cancers (6.4% vs 3.7%); (ii) a larger proportion of patients had disease detected by mammography (47% vs 10%); (iii) conservative breast excision was the only definitive surgery in 39% of cases (vs approximately 30%); and (iv) the mean size of lesions is smaller (13.5 mm vs 24.4 mm). Histologically, 26% of tumours were high grade, 71% had necrosis while 32% were oestrogen receptor (ER) and progesterone receptor (PR) negative. High grade tumours were associated with the presence of necrosis (P = 0.018), ER negativity (P = 0.015) and PR negativity (P = 0.001). CONCLUSIONS: This study reveals interesting trends of DCIS in Singapore. The sizeable proportion of hormone receptor-negative tumours may have implications for the hormonal adjuvant therapy of DCIS.