Tofacitinib inhibits the development of experimental autoimmune uveitis and reduces the proportions of Th1 but not of Th17 cells.

Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

TMP778, a selective inhibitor of RORγt, suppresses experimental autoimmune uveitis development, but affects both Th17 and Th1 cell populations.

Experimental autoimmune uveitis (EAU), an animal model for severe intraocular inflammatory eye diseases, is mediated by both Th1 and Th17 cells. Here, we examined the capacity of TMP778, a selective inhibitor of RORγt, to inhibit the development of EAU, as well as the related immune responses. EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). Treatment with TMP778 significantly inhibited the development of EAU, determined by histological examination. In addition, the treatment suppressed the cellular immune response to IRBP, determined by reduced production of IL-17 and IFN-γ, as well as lower percentages of lymphocytes expressing these cytokines, as compared to vehicle-treated controls. The inhibition of IFN-γ expression by TMP778 is unexpected in view of this compound being a selective inhibitor of RORγt. The observation was further confirmed by the finding of reduced expression of the T-bet (Tbx21) gene, the transcription factor for IFN-γ, by cells of TMP778-treated mice. Thus, these data demonstrate the capacity of TMP778 to inhibit pathogenic autoimmunity in the eye and shed new light on its mode of action in vivo.

MetaBridge: enabling network-based integrative analysis via direct protein interactors of metabolites.

Summary: Here, we present MetaBridge, a tool that collates protein interactors (curated metabolite-enzyme interactions) that influence the levels of specific metabolites including both biosynthetic and degradative enzymes. This enables network-based integrative analysis of metabolomics data with other omics data types. MetaBridge is designed to complement a systems-biology approach to analysis, pairing well with network analysis tools such as NetworkAnalyst.ca, but can be used in any bioinformatics workflow. Availability and implementation: MetaBridge has been implemented as a web tool at https://www.metabridge.org, and the source code is available at https://github.com/samhinshaw/metabridge_shiny (GNU GPLv3).

Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC.

Regulatory T-cells (Tregs) are responsible for homeostasis of the immune system, as well as for inhibition of pathogenic autoimmune processes. Induced-(i)-Tregs, can be generated in vitro by activation of CD4 cells in the presence of TGF-ß. A commonly used activation mechanism is by antibodies against CD3 and CD28. The physiological-like activation of T-cells, however, is with the specific target antigen presented by antigen-presenting cells (APC). The two modes of activation have been considered to yield the same populations of iTregs. Here, we compared between iTreg populations generated by either one of the two methods and found differences between their capacities to inhibit T-lymphocyte proliferative response, their expression of cell surface antigens and particularly, in their transcript expression profiles of certain chemokines and chemokine receptors. Our data thus indicate that iTregs generated by activation with anti-CD3/CD28 antibodies cannot be considered identical to iTregs generated by antigen/APC.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Dynamic molecular changes during the first week of human life follow a robust developmental trajectory.

Systems biology can unravel complex biology but has not been extensively applied to human newborns, a group highly vulnerable to a wide range of diseases. We optimized methods to extract transcriptomic, proteomic, metabolomic, cytokine/chemokine, and single cell immune phenotyping data from <1 ml of blood, a volume readily obtained from newborns. Indexing to baseline and applying innovative integrative computational methods reveals dramatic changes along a remarkably stable developmental trajectory over the first week of life. This is most evident in changes of interferon and complement pathways, as well as neutrophil-associated signaling. Validated across two independent cohorts of newborns from West Africa and Australasia, a robust and common trajectory emerges, suggesting a purposeful rather than random developmental path. Systems biology and innovative data integration can provide fresh insights into the molecular ontogeny of the first week of life, a dynamic developmental phase that is key for health and disease.

Digoxin Inhibits Induction of Experimental Autoimmune Uveitis in Mice, but Causes Severe Retinal Degeneration.

PURPOSE: Digoxin, a major medication for heart disease, was recently reported to have immunosuppressive capacity. Here, we determined the immunosuppressive capacity of digoxin on the development of experimental autoimmune uveitis (EAU) and on related immune responses. METHODS: The B10.A mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) and were treated daily with digoxin or vehicle control. On postimmunization day 14, the mouse eyes were examined histologically, while spleen cells were tested for cytokine production in response to IRBP and purified protein derivative. The immunosuppressive activity of digoxin was also tested in vitro, by its capacity to inhibit development of Th1 or Th17 cells. To investigate the degenerative effect of digoxin on the retina, naïve (FVB/N × B10.BR)F1 mice were similarly treated with digoxin and tested histologically and by ERG. RESULTS: Treatment with digoxin inhibited the development of EAU, as well as the cellular response to IRBP. Unexpectedly, treatment with digoxin suppressed the production of interferon-γ to a larger extent than the production of interleukin 17. Importantly, digoxin treatment induced severe retinal degeneration, determined by histologic analysis with thinning across all layers of the retina. Digoxin treatment also induced dose-dependent vision loss monitored by ERG on naïve mice without induction of EAU. CONCLUSIONS: Treatment of mice with digoxin inhibited the development of EAU and cellular immune response to IRBP. However, the treatment induced severe damage to the retina. Thus, the use of digoxin in humans should be avoided due to its toxicity to the retina.

Inflammasomes Induced by 7-Ketocholesterol and Other Stimuli in RPE and in Bone Marrow-Derived Cells Differ Markedly in Their Production of IL-1ß and IL-18.

PURPOSE: The inflammatory process plays a major role in the pathogenesis of AMD, and recent data indicate the involvement of inflammasomes. Inflammasomes are intracellular structures that trigger inflammation by producing mature interleukin-(IL)-1ß and IL-18. This study examined the capacity of 7-ketocholesterol (7KCh), an oxysterol that accumulates in the retinal pigmented epithelium (RPE) and choroid, to initiate inflammasome formation in RPE and bone marrow-derived cells. METHODS: Tested cells included fetal human RPE (fhRPE), human ARPE-19 cells, primary human brain microglia cells, and human THP-1 monocyte cells. 7-Ketocholesterol and other compounds were added to the cell cultures, and their stimulatory effects were determined by quantitative PCR and release of cytokines, measured by ELISA and Western blotting. RESULTS: 7-Ketocholesterol efficiently induced inflammasome formation by all primed cell populations, but secreted cytokine levels were higher in cultures of bone marrow-derived cells (microglia and THP-1 cells) than in RPE cultures. Interestingly, inflammasomes formed in cells of the two populations differed strikingly in their preferential production of the two cytokines. Thus, whereas bone marrow-derived cells produced levels of IL-1ß that were higher than those of IL-18, the opposite was found with RPE cells, which secreted higher levels of IL-18. Importantly, Western blot analysis showed that IL-18, but not IL-1ß, was expressed constitutively by RPE cells. CONCLUSIONS: 7-Ketocholesterol efficiently stimulates inflammasome formation and is conceivably involved in the pathogenesis of AMD. In contrast to bone marrow-derived cells, RPE cells produced higher levels of IL-18 than IL-1ß. Further, IL-18, a multifunctional cytokine, was expressed constitutively by RPE cells. These observations provide new information about stimuli and cells and their products assumed to be involved in the pathogenesis of AMD.

Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response.

BACKGROUND: Mutations in LRRK2 are related to certain forms of Parkinson's disease and, possibly, to the pathogenesis of Crohn's disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. METHODS: Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). RESULTS: The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. CONCLUSIONS: Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.

ITE, a novel endogenous nontoxic aryl hydrocarbon receptor ligand, efficiently suppresses EAU and T-cell-mediated immunity.

PURPOSE: Ligands for aryl hydrocarbon receptor (AHR), such as dioxins, are highly toxic. One such ligand, TCDD, was found to exert potent immunosuppressive capacities in mice developing pathogenic autoimmune processes, including EAU, but its toxicity makes it unusable for humans. A recently identified endogenous AHR ligand, ITE, is also immunosuppressive, but is nontoxic and could therefore be useful for therapy in humans. Here, we tested ITE for its capacity to inhibit EAU and related immune responses. METHODS: EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP; 40 µg) in CFA. Treatment with ITE was by daily intraperitoneal injection of 0.2 mg. Disease severity was assessed by both fundoscopy and histological examination. Draining lymph node cells were tested for proliferation by thymidine uptake and for cytokine production and release by ELISA. In addition, the intracellular expression of cytokines and Foxp3 was determined by flow cytometry. Serum antibodies were measured by ELISA. RESULTS: Treatment with ITE efficiently inhibited the development of EAU in mice, as well as the cellular immune responses against IRBP and PPD. ITE treatment inhibited the expansion of both Th1 and Th17 subpopulations, as well as their release of the signature cytokines, IFN-gamma and IL-17. The treatment moderately increased, however, the proportion of Foxp3 expressing T-regulatory cells. Antibody production was not affected by the treatment. CONCLUSIONS: ITE, an endogenous AHR ligand, efficiently inhibits EAU development and related cellular immune responses. Being nontoxic, ITE may be considered for treatment of pathogenic immunity in humans.