Long-Term Safety, Efficacy, and Patient-Centered Outcomes of Filgotinib in the Treatment of Rheumatoid Arthritis: Current Perspectives.

Filgotinib is an orally administered, preferential Janus kinase (JAK) inhibitor indicated for the treatment of moderate-to-severe rheumatoid arthritis (RA). The short-term safety, efficacy, and patient-reported outcomes (PROs) with filgotinib from Phase 2b/3 clinical trials (DARWIN 1 and 2; FINCH 1, 2, and 3) are described in patients who inadequately responded to methotrexate (MTX) and biologic disease-modifying antirheumatic drugs or who were naïve to MTX. This article reviews the safety and efficacy from the long-term extension (LTE) trials, DARWIN 3 (N=739) and FINCH 4 (N=2731), and PROs across the filgotinib development program in RA. Overall, in the DARWIN clinical trials (conducted from 2013-2023), patients received their LTE treatment for ≤8 years, while in the FINCH trials (ongoing from 2016-2025), patients received filgotinib treatment for ≤6 years in the LTE. The longer-term safety profile and consistent, sustained efficacy (American College of Rheumatology 20/50/70, Clinical Disease Activity Index, and Disease Activity Scale in 28 joints with C-reactive protein response rates) of filgotinib were largely similar to those observed in the shorter-term parent trials ≤52 weeks. PRO results from the parent trials showed improvements in patients' quality of life with filgotinib treatment, which compared to or exceeded improvements seen with placebo and active comparators (adalimumab, MTX). Filgotinib has a higher specificity for JAK1 compared with other therapeutic treatments, leading to reduced inhibition of JAK2/3-dependent pathways, potentially providing a distinct safety profile. Filgotinib is approved in Europe and Japan for treatment of people with moderate-to-severe RA, though it has not been approved by the US Food and Drug Administration, due to concerns around the benefit/risk profile of the filgotinib 200-mg dosage and the potential impact on semen parameters.

Filgotinib Modulates Inflammation-Associated Peripheral Blood Protein Biomarkers in Adults with Active Rheumatoid Arthritis and Prior Inadequate Response to Methotrexate.

INTRODUCTION: Our aim was to evaluate protein biomarker changes related to the administration of filgotinib, a Janus kinase (JAK) 1 preferential inhibitor, in patients with moderately to severely active rheumatoid arthritis (RA) with inadequate response to methotrexate. METHODS: Plasma and serum samples were collected from patients enrolled in FINCH 1 (NCT02889796), a Phase 3 trial. Patients with stable backgrounds of methotrexate were randomly assigned once-daily oral filgotinib 200 or 100 mg, subcutaneous adalimumab 40 mg every 2 weeks (W), or placebo. Up to 35 biomarkers were analyzed at baseline, W4, and W12 with enzyme-linked immunosorbent assays and chemiluminescence and electrochemiluminescence assays. RESULTS: At baseline, four distinct biomarker clusters were identified. The strongest intragroup correlations were in bone-cartilage resorption/inflammation and JAK/signal transducer and activator of transcription (STAT) signaling activity. At baseline, significant positive correlations were identified for cytokines with patient-reported pain and with patient measures of fatigue. Filgotinib reduced levels of cytokines associated with inflammation and cell migration as early as W4 and through W12. Compared to adalimumab, filgotinib induced significant reductions in bone-related turnover biomarkers, N-telopeptide of type 1 collagen and C-telopeptide 1, as well as biomarkers associated with baseline disease activity. No baseline predictors of therapeutic response to filgotinib were identified. CONCLUSIONS: Filgotinib reduced peripheral protein biomarkers associated with JAK/STAT signaling, inflammatory signaling, immune cell migration, and bone resorption as soon as W4 in FINCH 1. Effects were dose-dependent and consistent with the clinical efficacy of filgotinib observed in FINCH 1. The changes in peripheral biomarkers associated with filgotinib treatment in methotrexate-experienced patients are consistent with changes observed in both methotrexate-naïve and biologic disease-modifying antirheumatic drug-experienced RA populations. These data demonstrate dose-dependent effects of preferential JAK1 inhibition by filgotinib on peripheral blood protein biomarkers in methotrexate-experienced patients with RA. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02889796.

A sustainable wood biorefinery for low-carbon footprint chemicals production.

The profitability and sustainability of future biorefineries are dependent on efficient feedstock use. Therefore, it is essential to valorize lignin when using wood. We have developed an integrated biorefinery that converts 78 weight % (wt %) of birch into xylochemicals. Reductive catalytic fractionation of the wood produces a carbohydrate pulp amenable to bioethanol production and a lignin oil. After extraction of the lignin oil, the crude, unseparated mixture of phenolic monomers is catalytically funneled into 20 wt % of phenol and 9 wt % of propylene (on the basis of lignin weight) by gas-phase hydroprocessing and dealkylation; the residual phenolic oligomers (30 wt %) are used in printing ink as replacements for controversial para-nonylphenol. A techno-economic analysis predicts an economically competitive production process, and a life-cycle assessment estimates a lower carbon dioxide footprint relative to that of fossil-based production.

Neutrophil priming occurs in a sequential manner and can be visualized in living animals by monitoring IL-1ß promoter activation.

Rapid enhancement of phagocyte functionality is a hallmark of neutrophil priming. GeneChip analyses unveiled elevated CD54, dectin-2, and IL-1ß mRNA expression by neutrophils isolated from inflammatory sites. In fact, CD54 and dectin-2 protein expression was detected on neutrophils recovered from skin, peritoneal, and lung inflammation lesions but not on those in bone marrow or peripheral blood. Neutrophils increased CD54 and dectin-2 mRNA during migration in Boyden chambers and acquired CD54 and dectin-2 surface expression after subsequent exposure to GM-CSF. Neutrophils purified from IL-1ß promoter-driven DsRed-transgenic mice acquired DsRed signals during cell migration or exposure to GM-CSF. CD54 and dectin-2 were expressed by DsRed(+) (but not DsRed(-)) neutrophils in GM-CSF-supplemented cultures, and neutrophils recovered from inflammatory sites exhibited strong DsRed signals. The dynamic process of neutrophil priming was studied in chemically induced inflammatory skin lesions by monitoring DsRed expression using confocal microscopy. A majority (>80%) of Ly6G(+) neutrophils expressed DsRed, and those DsRed(+)/Ly6G(+) cells exhibited crawling motion with a higher velocity compared with their DsRed(-)/Ly6G(+) counterparts. This report unveils motile behaviors of primed neutrophils in living animals. We propose that neutrophil priming occurs in a sequential manner with rapid enhancement of phagocyte functionality, followed by CD54 and dectin-2 mRNA and protein expression, IL-1ß promoter activation, and accelerated motility. Not only do these findings provide a new conceptual framework for our understanding of the process of neutrophil priming, they also unveil new insights into the pathophysiology of many inflammatory disorders that are characterized by neutrophil infiltration.

Reciprocal regulation of development of neutrophil-dendritic cell hybrids in mice by IL-4 and interferon-gamma.

Neutrophils contribute to innate host immunity by functioning as professional phagocytes, whereas dendritic cells (DCs) are prototypic antigen presenting cells (APCs) responsible for the induction of adaptive immune responses. We have demonstrated recently that neutrophils trans-differentiate into a unique population, termed "neutrophil-DC hybrids," expressing surface markers of both neutrophils and DCs and exhibiting dual functionality of both phagocytes and APCs. Although the hybrid cells emerged in significant numbers in murine bone marrow (BM) culture in the presence of GM-CSF, mechanisms regulating their development remained mostly unknown. In this study, we tested a total of 61 cytokines for their potentials to regulate neutrophil-DC hybrid formation using a newly developed BM micro-culture system combined with semi-automated FACS analysis. Several cytokines including GM-CSF were found to promote the generation of neutrophil-DC hybrids defined by the phenotype of CD11c(+)/MHC II(+)/Ly6G(+). When tested in the presence of GM-CSF, hybrid cell development was enhanced by IL-4 and suppressed by interferon-γ (IFNγ) in dose-dependent fashions. We next determined in vivo impacts of IL-4 and IFNγ on the development of neutrophil-DC hybrids in thioglycollate-induced peritonitis lesions. Intraperitoneal administrations of IL-4/anti-IL-4 antibody complex (IL-4C) significantly increased the number of hybrids recovered from the lesions. By contract, recovery of hybrids was reduced by recombinant IFNγ. With regard to function, those hybrid cells recovered from IL-4C-treated mice and IFNγ-treated mice showed potent abilities to capture E.coli. These observations imply that emergence of neutrophil-DC hybrids in inflammatory sites is tightly regulated by local cytokine milieus.

Neutrophil differentiation into a unique hybrid population exhibiting dual phenotype and functionality of neutrophils and dendritic cells.

Neutrophils have been reported to acquire surface expression of MHC class II and co-stimulatory molecules as well as T-cell stimulatory activities when cultured with selected cytokines. However, cellular identity of those unusual neutrophils showing antigen presenting cell (APC)-like features still remains elusive. Here we show that both immature and mature neutrophils purified from mouse bone marrow differentiate into a previously unrecognized "hybrid" population showing dual properties of both neutrophils and dendritic cells (DCs) when cultured with granulocyte macrophage-colony-stimulating factor but not with other tested growth factors. The resulting hybrid cells express markers of both neutrophils (Ly6G, CXCR2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86). They also exhibit several properties typically reserved for DCs, including dendritic morphology, probing motion, podosome formation, production of interleukin-12 and other cytokines, and presentation of various forms of foreign protein antigens to naïve CD4 T cells. Importantly, they retain intrinsic abilities of neutrophils to capture exogenous material, extrude neutrophil extracellular traps, and kill bacteria via cathelicidin production. Not only do our results reinforce the notion that neutrophils can acquire APC-like properties, they also unveil a unique differentiation pathway of neutrophils into neutrophil-DC hybrids that can participate in both innate and adaptive immune responses.

Emergence, origin, and function of neutrophil-dendritic cell hybrids in experimentally induced inflammatory lesions in mice.

Although unusual neutrophils expressing major histocompatibility complex class II (MHC II) and costimulatory molecules have been detected at inflammatory sites in mice and humans, their identity, origin, and function remain unclear. We have demonstrated that, when cultured with granulocyte macrophage-colony-stimulating factor, neutrophils can give rise to a unique hybrid population exhibiting dual phenotypic and functionality of neutrophils and dendritic cells (DCs). Here we report that hybrid cells expressing surface markers of neutrophils (Ly6G, L-selectin, CXC chemokines receptor 2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86) become detectable in the peritoneal cavity, skin, lung, and lymph nodes under inflammatory conditions. Importantly, 20% to 30% of the adoptively transferred neutrophils acquired CD11c and MHC II expression when recovered from inflammatory lesions, demonstrating neutrophil → hybrid conversion in living animals. Using Escherichia coli strains expressing green fluorescent protein and ovalbumin, we further show hybrids play dual protective roles by rapidly clearing bacteria and presenting bacterial antigens to CD4 T cells. These results indicate that some of the neutrophils recruited to inflammatory lesions can differentiate into neutrophil-DC hybrids, thus challenging the classic view of neutrophils as terminally differentiated leukocytes destined to die or to participate primarily in host innate immunity.

Neutrophil-derived IL-1ß is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1ß/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1ß at the site of infection. Furthermore, neutrophil-derived IL-1ß was essential for host defense since adoptive transfer of IL-1ß-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1ß-deficient mice. S. aureus-induced IL-1ß production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1ß and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1ß production by neutrophils.

Real-time visualization of macromolecule uptake by epidermal Langerhans cells in living animals.

As a skin-resident member of the dendritic cell family, Langerhans cells (LCs) are generally regarded to function as professional antigen-presenting cells. Here we report a simple method to visualize the endocytotic activity of LCs in living animals. BALB/c mice received subcutaneous injection of FITC-conjugated dextran (DX) probes into the ear skin and were then examined under confocal microscopy. Large numbers of FITC(+) epidermal cells became detectable 12-24 hours after injection as background fluorescence signals began to disappear. Most (>90%) of the FITC(+) epidermal cells expressed Langerin, and >95% of Langerin(+) epidermal cells exhibited significant FITC signals. To assess intracellular localization, Alexa Fluor 546-conjugated DX probes were locally injected into IAß-enhanced green fluorescent protein (EGFP) knock-in mice and Langerin-EGFP-diphtheria toxin receptor mice--three dimensional rotation images showed close association of most of the internalized DX probes with major histocompatibility complex (MHC) class II molecules, but not with Langerin molecules. These observations support the current view that LCs constantly sample surrounding materials, including harmful and innocuous antigens, at the environmental interface. Our data also validate the potential utility of the newly developed imaging approach to monitor LC function in wild-type animals.

Intravital imaging of IL-1beta production in skin.

IL-1 is a prototypic inflammatory cytokine that has pathogenic roles in various skin disorders. Although Langerhans cells (LCs) have been reported to express IL-1beta mRNA upon application of contact sensitizers, it remains unclear whether other cell types produce IL-1beta in skin. Thus, we sought to directly identify IL-1beta-producing cells in living animals by construction of transgenic mice expressing DsRed fluorescence protein gene under the control of IL-1beta promoter. Little DsRed fluorescence signal was detected in skin under steady-state conditions. Striking increases in DsRed signal were observed after topical application of a contact sensitizer, oxazolone, which also induced markedly elevated IL-1beta mRNA and protein expression. DsRed signal was expressed primarily by CD45(+)/CD11b(+) myeloid leukocytes in both epidermal and dermal compartments and was detected only in small fractions of epidermal LCs. Interestingly, DsRed(+) cells emerged preferentially as clusters around hair follicles. Intravital confocal imaging experiments revealed highly motile potentials of DsRed(+) cells-they constantly crawled around hair follicles via amoeba-like movements with a mean velocity of 1.0+/-0.4 microm min(-1) (epidermis) or 2.7+/-1.4 microm min(-1) (dermis). The newly developed in vivo imaging system represents a useful tool for studying spatial regulation of IL-1beta production in skin.

Bidirectional homing of Tregs between the skin and lymph nodes.

Although several homing receptors are known to be differentially expressed by Tregs in lymphoid tissues compared with those found in peripheral tissues, it remains unclear whether these cells traffic between the two locations. In this issue of the JCI, Tomura et al. report steady-state Treg migration from the skin to draining LNs in mice. Furthermore, they report that not only does skin inflammation exacerbate LN-directed Treg homing, it also triggers reverse circulation of Tregs from LNs to skin, whereby these cells contribute to regulation of the immune response. These results now form a new framework for our understanding of Treg homing.

IL-33 promotes DC development in BM culture by triggering GM-CSF production.

Short-term DC cultures generated with GM-CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c+ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL-33 was found to augment DC development time- and dose-dependently. Although the resulting CD11c+ cells generated in the presence of IL-33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL-12 p70, displayed PD-L1 and PD-L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL-33-induced expansion of CD11c+ cells was completely blocked by anti-GM-CSF mAb, and GM-CSF mRNA and protein expression in BM culture was markedly elevated by added IL-33, indicating that IL-33 promotes in vitro DC generation indirectly by a GM-CSF-dependent manner. With regard to the cellular source, IL-33-dependent GM-CSF production was observed exclusively within the CD45+/FcepsilonRI+ BM population. Not only do our results reinforce the notion that GM-CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.

Dual therapeutic efficacy of vinblastine as a unique chemotherapeutic agent capable of inducing dendritic cell maturation.

Our recent unbiased functional screen of 54 chemotherapeutic drugs unveiled striking heterogeneity in their effects on dendritic cells (DC). Most notably, vinblastine (VBL) was found to induce phenotypic and functional maturation of DCs in vitro. Here, we sought to determine whether VBL exhibits "dual" therapeutic efficacy in living animals by directly killing tumor cells and by boosting host immunity via DC maturation. Local injection of VBL in a low dose into the skin of C57BL/6 mice induced in situ maturation of epidermal Langerhans cells. When coinjected with a model antigen, ovalbumin (OVA), VBL enhanced OVA-specific cellular and humoral immune responses. When injected directly into the OVA cDNA-transduced E.G7 tumors, VBL augmented clonal expansion of OVA-reactive CD8 T cells and CTL activities. In B16 melanoma model, intratumor VBL injection induced apoptosis of melanoma cells, phenotypic maturation of tumor-infiltrating DCs, and significant CTL activities. Although complete clearance was never achieved, growth kinetic of B16 melanoma was markedly reduced in C57BL/6 mice by intratumor VBL injection. Importantly, the same treatment was far less efficacious in immunocompromised severe combined immunodeficient mice, indicating the requirement of intact host immunity. Our results introduce a new concept that VBL may be used to design "immunostimulatory" chemotherapy regimens.

Classification of chemotherapeutic agents based on their differential in vitro effects on dendritic cells.

Despite the crucial roles dendritic cells (DC) play in host immunity against cancer, the pharmacologic effects of many chemotherapeutic agents have remained mostly unknown. We recently developed a DC biosensor clone by engineering the stable murine DC line XS106 to express the yellow fluorescent protein (YFP) gene under the control of interleukin (IL)-1beta promoter. In this study, the resulting XS106 pIL1-YFP DC clone was used to screen 54 anticancer drugs. Each drug was tested at five concentrations (0.1-10 micromol/L) for its effects on YFP expression, cell viability, and granulocyte macrophage colony-stimulating factor-dependent growth. Our unbiased systematic screening unveiled a striking heterogeneity among the tested anticancer drugs in their effects on the three functional variables. Interestingly, 15 drugs induced significant YFP expression at subcytotoxic concentrations and were thus categorized as "DC-stimulatory" anticancer drugs. These drugs were subsequently found to induce at least one of the characteristic maturational changes in mouse bone marrow-derived DCs. For example, vinblastine, a prototypic drug of this class, induced the production of IL-1beta, IL-6, and IL-12, increased surface expression of CD40, CD80, CD86, and MHC class II, and augmented T cell-stimulatory capacity of DCs. Not only do these results illustrate the differential pharmacologic effects of commonly used chemotherapeutic agents on DCs, they may also provide a conceptual framework for rationale-based selection and combination of anticancer drugs for clinical application.

Identification of crassin acetate as a new immunosuppressant triggering heme oxygenase-1 expression in dendritic cells.

By screening 720 natural compounds in a standard 2-way allogeneic mixed leukocyte reaction assay, we identified a potent immunosuppressive capacity of crassin acetate (CRA), a coral-derived cembrane diterpenoid. CRA efficiently inhibited allogeneic mixed leukocyte reaction as well as antigen-specific activation of CD4 T cells by bone marrow-derived dendritic cells (DCs). With regard to cellular targets, CRA suppressed not only mitogen-triggered T-cell activation, but also lipopolysaccharide-induced DC maturation, indicating dual functionality. Treatment with CRA at nontoxic doses induced heme oxygenase-1 (HO-1) mRNA/protein expression and HO-1 enzymatic activity in DCs, suggesting a unique mechanism of action. In fact, lipopolysaccharide-induced DC maturation was also inhibited by structurally unrelated compounds known to induce HO-1 expression or carbon monoxide (CO) release. Allergic contact hypersensitivity response to oxazolone and oxazolone-induced Langerhans cell migration from epidermis were both prevented almost completely by systemic administration of CRA. Not only do our results support the recent concept that HO-1/CO system negatively regulates immune responses, they also form both conceptual and technical frameworks for a more systematic, large-scale drug discovery effort to identify HO-1/CO-targeted immunosuppressants with dual target specificity.

Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells.

Osteopontin (OPN), originally discovered in bone as an extracellular matrix protein, was identified in many cell types in the immune system, presumably being involved in many aspects of pathogenesis of inflammatory and immune diseases. Mast cells are also involved in such pathological aspects by secreting multiple mediators. However, it has not been determined whether mast cells produce OPN and whether it affects their function. To test this, we used murine fetal skin-derived cultured mast cells (FSMC) and bone marrow-derived cultured mast cells. We found that OPN was spontaneously produced by FSMC and inducible by ionomycin and FcepsilonRI aggregation in bone marrow-derived cultured mast cells. In the presence of mast cell growth factors, FSMC were similarly generated from both OPN-deficient (OPN(-/-)) and -sufficient (OPN(+/+)) mice without significant differences in yield, purity, granularity, and viability. Using OPN(-/-) FSMC, we found that recombinant OPN augmented IgE-mediated degranulation and induced FSMC chemotaxis. Both effects were mediated by OPN receptors (i.e. CD44 and integrin alphav). IgE-mediated passive cutaneous anaphylaxis was significantly reduced in OPN(-/-) mice compared with OPN(+/+) mice, indicating physiological relevance of OPN. These results indicate that OPN is a mast cell mediator, enhances mast cell responses to antigen, and thus may influence mast cell-related pathological conditions.

Identification of novel pharmacological activities of an antifungal agent, nystatin, to promote dendritic cell maturation.

As an unbiased functional screen to identify agents activating dendritic cells (DCs), we recently developed a DC-based biosensor system, in which a stable murine DC line XS106 was engineered to express the yellow fluorescent protein (YFP) gene under the control of the IL-1beta promoter. Here we report that nystatin (NYT), an antifungal drug of the family of polyene macrolide antibiotics, elevated YFP expression by the resulting XS106-pIL1-YFP DC biosensor clone in a dose-dependent fashion. With respect to the underlying mechanisms, NYT activated the NFkappaB p65 and c-Rel subunits in the parental XS106 DC line. Moreover, NYT dose-dependently increased the surface expression of major histocompatibility complex (MHC) class II (MHC II), CD40, CD54, CD80, and CD86 by murine bone marrow-derived DCs and triggered their robust production of IL-1beta, IL-6, IL-12, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha. Our results document previously unrecognized pharmacological activities of the most commonly used antifungal drug to promote DC maturation.