Baricitinib-associated changes in global gene expression during a 24-week phase II clinical systemic lupus erythematosus trial implicates a mechanism of action through multiple immune-related pathways.

OBJECTIVE: To characterise the molecular pathways impacted by the pharmacologic effects of the Janus kinase (JAK) 1 and JAK2 inhibitor baricitinib in SLE. METHODS: In a phase II, 24-week, randomised, placebo-controlled, double-blind study (JAHH), RNA was isolated from whole blood in 274 patients and analysed using Affymetrix HTA2.0 array. Serum cytokines were measured using ultrasensitive quantitative assays. RESULTS: Gene expression profiling demonstrated an elevation of STAT1, STAT2 and multiple interferon (IFN) responsive genes at baseline in patients with SLE. Statistical and gene network analyses demonstrated that baricitinib treatment reduced the mRNA expression of functionally interconnected genes involved in SLE including STAT1-target, STAT2-target and STAT4-target genes and multiple IFN responsive genes. At baseline, serum cytokines IFN-α, IFN-γ, interleukin (IL)-12p40 and IL-6 were measurable and elevated above healthy controls. Treatment with baricitinib significantly decreased serum IL-12p40 and IL-6 cytokine levels at week 12, which persisted through week 24. CONCLUSION: Baricitinib treatment induced significant reduction in the RNA expression of a network of genes associated with the JAK/STAT pathway, cytokine signalling and SLE pathogenesis. Baricitinib consistently reduced serum levels of two key cytokines implicated in SLE pathogenesis, IL-12p40 and IL-6.

Infections in baricitinib clinical trials for patients with active rheumatoid arthritis.

OBJECTIVES: To evaluate the incidence of infection in patients with active rheumatoid arthritis (RA) treated with baricitinib, an oral selective Janus kinase (JAK)1 and JAK2 inhibitor. METHODS: Infections are summarised from an integrated database (8 phase 3/2/1b clinical trials and 1 long-term extension (LTE)) with data to 1 April 2017. The 'all-bari-RA' analysis set included patients who received any baricitinib dose. Placebo comparison was based on six studies with 4 mg and placebo to week 24, including four trials with 2 mg (placebo-controlled set). Dose-response assessment was based on four studies with 2 mg and 4 mg, including LTE data (2-4 mg extended set). RESULTS: There were 3492 patients who received baricitinib for 7860 patient-years (PY) of exposure (median 2.6 years, maximum 6.1 years). Treatment-emergent infections were higher for baricitinib versus placebo (exposure-adjusted incidence rate (IR)/100 PY: placebo 75.9, 2 mg 84.0 (p not significant), 4 mg 88.4 (p≤0.001)). The IR of serious infection was similar for baricitinib versus placebo and stable over time (all-bari-RA IR 3.0/100 PY). There were 11 cases of tuberculosis (all-bari-RA IR 0.1/100 PY); all occurred with 4 mg in endemic regions. Herpes zoster (HZ) IR/100 PY was higher for baricitinib versus placebo (placebo 1.0, 2 mg 3.1 (p not significant), 4 mg 4.3 (p≤0.01)); rates remained elevated and stable over time (all-bari-RA 3.3). Opportunistic infections, including multidermatomal HZ, were infrequent in the baricitinib programme (all-bari-RA IR 0.5/100 PY). CONCLUSIONS: Increased rates of treatment-emergent infections including HZ were observed in patients with RA treated with baricitinib, consistent with baricitinib's immunomodulatory mode of action.

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.

Comparison of baricitinib, upadacitinib, and tofacitinib mediated regulation of cytokine signaling in human leukocyte subpopulations.

BACKGROUND: The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level. METHODS: Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC50) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC50 and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type. RESULTS: Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC50 values and increased time above IC50 translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees. CONCLUSIONS: Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF.

Characterization and Changes of Lymphocyte Subsets in Baricitinib-Treated Patients With Rheumatoid Arthritis: An Integrated Analysis.

OBJECTIVE: Baricitinib is an orally administered inhibitor of JAK1 and JAK2 that has been shown to be effective in treating rheumatoid arthritis (RA). This study was undertaken to analyze changes in lymphocyte cell subsets during baricitinib treatment and to correlate these changes with clinical outcomes. METHODS: An integrated analysis was conducted by pooling data from 3 completed phase III trials comparing placebo with baricitinib treatment (RA-BEAM, RA-BUILD, and RA-BEACON) and 1 ongoing long-term extension study (RA-BEYOND) in patients with active RA (n = 2,186). RESULTS: Baricitinib treatment was associated with an early transient increase in total lymphocyte count at week 4, which returned to baseline by week 12. Transient changes within normal reference ranges in T cells and subsets were observed with baricitinib treatment, up to week 104. B cells and relevant subpopulations increased after 4 weeks of baricitinib treatment, with no further increases noted through 104 weeks of treatment. Natural killer (NK) cells temporarily increased after 4 weeks of baricitinib treatment, before decreasing below baseline levels and then stabilizing over time. With baricitinib treatment, few correlations were observed between changes in lymphocyte subsets and clinical end points, and most correlations were also observed within the placebo group. A modest potential association between low NK cell numbers and treatment-emergent infections was observed in the baricitinib 4 mg/day treatment group, but not for serious infections or herpes zoster. CONCLUSION: Overall, these findings demonstrate that changes in lymphocyte subsets were largely within normal reference ranges across the baricitinib phase III RA clinical program and were not associated with increased risk of serious infections.

Topical Prostaglandin E Analog Restores Defective Dendritic Cell-Mediated Th17 Host Defense Against Methicillin-Resistant Staphylococcus Aureus in the Skin of Diabetic Mice.

People with diabetes are more prone to Staphylococcus aureus skin infection than healthy individuals. Control of S. aureus infection depends on dendritic cell (DC)-induced T-helper 17 (Th17)-mediated neutrophil recruitment and bacterial clearance. DC ingestion of infected apoptotic cells (IACs) drive prostaglandin E2 (PGE2) secretion to generate Th17 cells. We speculated that hyperglycemia inhibits skin DC migration to the lymph nodes and impairs the Th17 differentiation that accounts for poor skin host defense in diabetic mice. Diabetic mice showed increased skin lesion size and bacterial load and decreased PGE2 secretion and Th17 cells compared with nondiabetic mice after methicillin-resistant S. aureus (MRSA) infection. Bone marrow-derived DCs (BMDCs) cultured in high glucose (25 mmol/L) exhibited decreased Ptges mRNA expression, PGE2 production, lower CCR7-dependent DC migration, and diminished maturation after recognition of MRSA-IACs than BMDCs cultured in low glucose (5 mmol/L). Similar events were observed in DCs from diabetic mice infected with MRSA. Topical treatment of diabetic mice with the PGE analog misoprostol improved host defense against MRSA skin infection by restoring DC migration to draining lymph nodes, Th17 differentiation, and increased antimicrobial peptide expression. These findings identify a novel mechanism involved in poor skin host defense in diabetes and propose a targeted strategy to restore skin host defense in diabetes.

TH9 cells are required for tissue mast cell accumulation during allergic inflammation.

BACKGROUND: IL-9 is important for the growth and survival of mast cells. IL-9 is produced by T cells, natural killer T cells, mast cells, eosinophils, and innate lymphoid cells, although the cells required for mast cell accumulation during allergic inflammation remain undefined. OBJECTIVE: We sought to elucidate the role of TH9 cells in promoting mast cell accumulation in models of allergic lung inflammation. METHODS: Adoptive transfer of ovalbumin-specific TH2 and TH9 cells was used to assess the ability of each subset to mediate mast cell accumulation in tissues. Mast cell accumulation was assessed in wild-type mice and mice with PU.1-deficient T cells subjected to acute and chronic models of allergic inflammation. RESULTS: Adoptive transfer experiments demonstrated that recipients of TH9 cells had significantly higher mast cell accumulation and expression of mast cell proteases compared with control or TH2 recipients. Mast cell accumulation was dependent on IL-9, but not IL-13, a cytokine required for many aspects of allergic inflammation. In models of acute and chronic allergic inflammation, decreased IL-9 levels in mice with PU.1-deficient T cells corresponded to diminished tissue mast cell numbers and expression of mast cell proteases. Mice with PU.1-deficient T cells have defects in IL-9 production from CD4(+) T cells, but not natural killer T cells or innate lymphoid cells, suggesting a TH cell-dependent phenotype. Rag1(-/-) mice subjected to a chronic model of allergic inflammation displayed reduced mast cell infiltration comparable with accumulation in mice with PU.1-deficient T cells, emphasizing the importance of IL-9 produced by T cells in mast cell recruitment. CONCLUSION: TH9 cells are a major source of IL-9 in models of allergic inflammation and play an important role in mast cell accumulation and activation.

Th17 cells demonstrate stable cytokine production in a proallergic environment.

Th17 cells are critical for the clearance of extracellular bacteria and fungi, but also contribute to the pathology of autoimmune diseases and allergic inflammation. After exposure to an appropriate cytokine environment, Th17 cells can acquire a Th1-like phenotype, but less is known about their ability to adopt Th2 and Th9 effector programs. To explore this in more detail, we used an IL-17F lineage tracer mouse strain that allows tracking of cells that formerly expressed IL-17F. In vitro-derived Th17 cells adopted signature cytokine and transcription factor expression when cultured under Th1-, Th2-, or Th9-polarizing conditions. In contrast, using two models of allergic airway disease, Th17 cells from the lungs of diseased mice did not adopt Th1, Th2, or Th9 effector programs, but remained stable IL-17 secretors. Although in vitro-derived Th17 cells expressed IL-4Rα, those induced in vivo during allergic airway disease did not, possibly rendering them unresponsive to IL-4-induced signals. However, in vitro-derived, Ag-specific Th17 cells transferred in vivo to OVA and aluminum hydroxide-sensitized mice also maintained IL-17 secretion and did not produce alternative cytokines upon subsequent OVA challenge. Thus, although Th17 cells can adopt new phenotypes in response to some inflammatory environments, our data suggest that in allergic inflammation, Th17 cells are comparatively stable and retain the potential to produce IL-17. This might reflect a cytokine environment that promotes Th17 stability, and allow a broader immune response at tissue barriers that are susceptible to allergic inflammation.

STAT4 is required for IL-23 responsiveness in Th17 memory cells and NKT cells.

STAT4 is a critical mediator of inflammatory immunity and is required for all known IL-12 biological responses, including the induction of IFN-γ and development of Th1 cells. We demonstrate that IL-23, an IL-12-related cytokine, also requires STAT4 for optimal IL-17 secretion from memory T helper cells and NKT cells. Although IL-23 stimulation had modest effects on STAT4 activation, STAT4-deficiency results in reduced Il23r expression. These data demonstrate a restricted requirement for STAT4 in innate and adaptive IL-17-secreting T cell responses that might contribute to inflammatory immunity.