Lupus-Associated Immune Complexes Activate Human Neutrophils in an FcγRIIA-Dependent but TLR-Independent Response.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nucleic acids and nucleoproteins. Anti-dsDNA Abs are considered a hallmark of SLE, and previous studies have indicated that nucleic acid-containing immune complexes (ICs) induce B cell and dendritic cell activation in a TLR-dependent process. How ICs containing nucleic acids affect neutrophil function has not been well investigated. In this study, we report that nucleic acid-containing ICs derived from the sera of SLE patients induce human and mouse neutrophil activation through TLR-independent mechanisms. Soluble ICs containing Sm/RNP, an RNA Ag, activate human neutrophils to produce reactive oxygen species (ROS) and IL-8. In contrast, ICs containing DNA have to be immobilized to efficiently activate neutrophils. We found that deleting TLR7 or TLR9, the receptors for RNA and DNA, had no effect on mouse neutrophil activation induced by RNA-containing and immobilized DNA-containing ICs. Binding of ICs are mediated through FcγRIIA and FcγRIIIB. However, neutrophil activation induced by RNA- and DNA-containing ICs requires FcγRIIA, as blocking FcγRIIA inhibited ROS release from neutrophils. RNA-containing ICs induce calcium flux, whereas TLR7/8 ligand R848 do not. Surprisingly, chloroquine inhibits calcium flux induced by RNA-containing ICs, suggesting that this lesser known function of chloroquine is involved in the neutrophil activation induced by ICs. These data indicate the SLE-derived ICs activate neutrophils to release ROS and chemokines in an FcγRIIA-dependent and TLR7- and TLR9-independent manner that likely contributes to local tissue inflammation and damage.

Pilot Study of Low-dose Naltrexone for the Treatment of Chronic Pain Due to Arthritis: A Randomized, Double-blind, Placebo-controlled, Crossover Clinical Trial.

PURPOSE: Low-dose naltrexone (LDN) is commonly used to control pain and other symptoms, especially in patients with autoimmune diseases, but with limited evidence. This study tests the efficacy of LDN in reducing chronic pain in patients with osteoarthritis (OA) and inflammatory arthritis (IA), where existing approaches often fail to adequately control pain. METHODS: In this randomized, double-blind, placebo-controlled, crossover clinical trial, each patient received 4.5 mg LDN for 8 weeks and placebo for 8 weeks. Outcome measures were patient reported, using validated questionnaires. The primary outcome was differences in pain interference during the LDN and placebo periods, using the Brief Pain Inventory (scale, 0-70). Secondary outcomes included changes in mean pain severity, fatigue, depression, and multiple domains of health-related quality of life. The painDETECT questionnaire classified pain as nociceptive, neuropathic, or mixed. Data were analyzed using mixed-effects models. FINDINGS: Seventeen patients with OA and 6 with IA completed the pilot study. Most patients described their pain as nociceptive (n = 9) or mixed (n = 8) rather than neuropathic (n = 3). There was no difference in change in pain interference after treatment with LDN (mean [SD], -23 [19.4]) versus placebo (mean [SD], -22 [19.2]; P = 0.90). No significant differences were seen in pain severity, fatigue, depression, or health-related quality of life. IMPLICATIONS: In this small pilot study, findings do not support LDN being efficacious in reducing nociceptive pain due to arthritis. Too few patients were enrolled to rule out modest benefit or to assess inflammatory or neuropathic pain. CLINICALTRIALS: gov identifier: NCT03008590.

The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells.

Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.

A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

OBJECTIVE: A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. RESULTS: In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. CONCLUSIONS: We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy.

Type 1 IFN mediates cross-talk between innate and adaptive immunity that abrogates transplantation tolerance.

TLR activation of innate immunity prevents the induction of transplantation tolerance and shortens skin allograft survival in mice treated with costimulation blockade. The mechanism by which TLR signaling mediates this effect has not been clear. We now report that administration of the TLR agonists LPS (TLR4) or polyinosinic:polycytidylic acid (TLR3) to mice treated with costimulation blockade prevents alloreactive CD8(+) T cell deletion, primes alloreactive CTLs, and shortens allograft survival. The TLR4- and MyD88-dependent pathways are required for LPS to shorten allograft survival, whereas polyinosinic:polycytidylic acid mediates its effects through a TLR3-independent pathway. These effects are all mediated by signaling through the type 1 IFN (IFN-alphabeta) receptor. Administration of IFN-beta recapitulates the detrimental effects of TLR agonists on transplantation tolerance. We conclude that the type 1 IFN generated as part of an innate immune response to TLR activation can in turn activate adaptive immune responses that abrogate transplantation tolerance. Blocking of type 1 IFN-dependent pathways in patients may improve allograft survival in the presence of exogenous TLR ligands.

B cells and dendritic cells from V kappa 8 light chain transgenic mice activate MRL-lpr/gld CD4+ T cells.

Autoreactive CD4+ T cells are required for full expression of disease in human systemic lupus erythematosus and in spontaneous murine lupus. However, the Ag specificity of these CD4+ T cells remains largely unknown. Rheumatoid factor (RF) B cells function as highly efficient APCs by taking up immune complexes (IC) and presenting IC constituents to T cells. We hypothesized that Ag-specific CD4+ T cells in lupus-prone mice could be identified by stimulating the CD4+ T cells with RF B cells from AM14 RF BCR transgenic mice pulsed with IC containing lupus-associated autoantibodies and autoantigens. This approach identified several independent T cell lines that proliferated robustly in response to IC-pulsed spleen cells from the AM14 RF BCR transgenic mice. However, these T cells did not recognize an IC constituent. Instead, these T cells recognized a determinant dependent on the inheritance of the transgene-encoded Vkappa8 L chain, most likely a neoantigen created by the insertion of the transgene into the genome. Additionally, although the precise nature of the neoantigen is not known, the T cells described in this report may provide a useful tool for examining the role of T cells in the RF autoantibody response.