Modulation of Disease-Associated Pathways in Hidradenitis Suppurativa by the Janus Kinase 1 Inhibitor Povorcitinib: Transcriptomic and Proteomic Analyses of Two Phase 2 Studies.

Janus kinase (JAK)/signal transducer and activator of transcription signaling (STAT) has been implicated in the pathophysiology of hidradenitis suppurativa (HS). This study evaluated treatment-related transcriptomic and proteomic changes in patients with moderate-to-severe HS treated with the investigational oral JAK1-selective inhibitor povorcitinib (INCB054707) in two phase 2 trials. Lesional skin punch biopsies (baseline and Week 8) were taken from active HS lesions of patients receiving povorcitinib (15 or 30 mg) once daily (QD) or a placebo. RNA-seq and gene set enrichment analyses were used to evaluate the effects of povorcitinib on differential gene expression among previously reported gene signatures from HS and wounded skin. The number of differentially expressed genes was the greatest in the 30 mg povorcitinib QD dose group, consistent with the published efficacy results. Notably, the genes impacted reflected JAK/STAT signaling transcripts downstream of TNF-α signaling, or those regulated by TGF-ß. Proteomic analyses were conducted on blood samples obtained at baseline and Weeks 4 and 8 from patients receiving povorcitinib (15, 30, 60, or 90 mg) QD or placebo. Povorcitinib was associated with transcriptomic downregulation of multiple HS and inflammatory signaling markers as well as the reversal of gene expression previously associated with HS lesional and wounded skin. Povorcitinib also demonstrated dose-dependent modulation of several proteins implicated in HS pathophysiology, with changes observed by Week 4. The reversal of HS lesional gene signatures and rapid, dose-dependent protein regulation highlight the potential of JAK1 inhibition to modulate underlying disease pathology in HS.

RNA Aptamer Delivery through Intact Human Skin.

It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.

Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans.

Tapinarof (GSK2894512) is a naturally derived topical treatment with demonstrated efficacy for patients with psoriasis and atopic dermatitis, although the biologic target and mechanism of action had been unknown. We demonstrate that the anti-inflammatory properties of tapinarof are mediated through activation of the aryl hydrocarbon receptor (AhR). We show that tapinarof binds and activates AhR in multiple cell types, including cells of the target tissue-human skin. In addition, tapinarof moderates proinflammatory cytokine expression in stimulated peripheral blood CD4+ T cells and ex vivo human skin, and impacts barrier gene expression in primary human keratinocytes; both of these processes are likely to be downstream of AhR activation based on current evidence. That the anti-inflammatory properties of tapinarof derive from AhR agonism is conclusively demonstrated using the mouse model of imiquimod-induced psoriasiform skin lesions. Topical treatment of AhR-sufficient mice with tapinarof leads to compound-driven reductions in erythema, epidermal thickening, and tissue cytokine levels. In contrast, tapinarof has no impact on imiquimod-induced skin inflammation in AhR-deficient mice. In summary, these studies identify tapinarof as an AhR agonist and confirm that its efficacy is dependent on AhR.

Act1: A Psoriasis Susceptibility Gene Playing its Part in Keratinocytes.

Unchecked inflammation, impaired keratinocyte differentiation, and heightened host defense responses typify psoriasis. Lambert et al. make clever use of psoriasis patient genetics and whole transcriptome RNA-Seq analysis to implicate Act1 in these seemingly variegated processes by keeping IL-17 receptor signaling in check while supporting differentiation and limiting innate immune responses in human keratinocytes.

Development of a Topical Treatment for Psoriasis Targeting RORγ: From Bench to Skin.

BACKGROUND: Psoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy. METHODS AND FINDINGS: The preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin. CONCLUSIONS: Our work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.

Altered subcellular localization of IL-33 leads to non-resolving lethal inflammation.

Non-resolving inflammation is a major contributor to chronic disease pathogenesis, including that of cancer, chronic obstructive pulmonary disease, asthma, arthritis, inflammatory bowel disease, multiple sclerosis and obesity. Some cytokines, such as IL-1α and IL-33, may act as endogenous alarmins that contribute to non-resolving inflammation. These cytokines are constitutively expressed in the nucleus and are thought to promote inflammation only upon release during tissue damage or cell necrosis. However, the importance of their nuclear localization in immune homeostasis is not fully understood. We describe herein a novel mouse model in which the nuclear localization signal of IL-33 is abolished and demonstrate for the first time that, alone, altered subcellular localization of IL-33 dramatically affects immune homeostasis. Heterozygous IL33(tm1/+) mice display elevated serum IL-33 levels, indicating that IL-33 is constitutively released when not actively targeted to the nucleus. IL33(tm1/+) mice succumb to lethal inflammation characterized by eosinophil-dominated immune cell infiltration of multiple organs. The profound inflammatory phenotype is dependent on mediators downstream of ST2 as ST2-null mice are protected in spite of high serum IL-33 levels. Importantly, IL-33 transcript levels in this knock-in mouse model remain under endogenous control. We adopt the term "nuclear alarmin" to describe a danger signal that is primarily regulated by nuclear compartmentalization in this fashion.

Peritoneal cavity regulatory B cells (B10 cells) modulate IFN-γ+CD4+ T cell numbers during colitis development in mice.

The spleen regulatory B cell subset with the functional capacity to express IL-10 (B10 cells) modulates both immune responses and autoimmune disease severity. However, the peritoneal cavity also contains relatively high frequencies of functionally defined IL-10-competent B10 cells. In this study, peritoneal cavity B10 cells shared similar cell surface phenotypes with their spleen counterparts. However, peritoneal cavity B10 cells were 10-fold more frequent among B cells than occurred within the spleen, intestinal tract, or mesenteric lymph nodes and were present at higher proportions among the phenotypically defined peritoneal B1a > B1b > B2 cell subpopulations. The development or localization of B10 cells within the peritoneal cavity was not dependent on the presence of commensal microbiota, T cells, IL-10 or B10 cell IL-10 production, or differences between their fetal liver or adult bone marrow progenitor cell origins. The BCR repertoire of peritoneal cavity B10 cells was diverse, as occurs in the spleen, and predominantly included germline-encoded VH and VL regions commonly found in either the conventional or B1 B cell compartments. Thereby, the capacity to produce IL-10 appears to be an intrinsic functional property acquired by clonally diverse B cells. Importantly, IL-10 production by peritoneal cavity B cells significantly reduced disease severity in spontaneous and induced models of colitis by regulating neutrophil infiltration, colitogenic CD4(+) T cell activation, and proinflammatory cytokine production during colitis onset. Thus, the numerically small B10 cell subset within the peritoneal cavity has regulatory function and is important for maintaining homeostasis within gastrointestinal tissues and the immune system.

Maternal stress and effects of prenatal air pollution on offspring mental health outcomes in mice.

BACKGROUND: Low socioeconomic status is consistently associated with reduced physical and mental health, but the mechanisms remain unclear. Increased levels of urban air pollutants interacting with parental stress have been proposed to explain health disparities in respiratory disease, but the impact of such interactions on mental health is unknown. OBJECTIVES: We aimed to determine whether prenatal air pollution exposure and stress during pregnancy act synergistically on offspring to induce a neuroinflammatory response and subsequent neurocognitive disorders in adulthood. METHODS: Mouse dams were intermittently exposed via oropharyngeal aspiration to diesel exhaust particles (DEP; 50 µg × 6 doses) or vehicle throughout gestation. This exposure was combined with standard housing or nest material restriction (NR; a novel model of maternal stress) during the last third of gestation. RESULTS: Adult (postnatal day 60) offspring of dams that experienced both stressors (DEP and NR) displayed increased anxiety, but only male offspring of this group had impaired cognition. Furthermore, maternal DEP exposure increased proinflammatory interleukin (IL)-1ß levels within the brains of adult males but not females, and maternal DEP and NR both decreased anti-inflammatory IL-10 in male, but not female, brains. Similarly, only DEP/NR males showed increased expression of the innate immune recognition gene toll-like receptor 4 (Tlr4) and its downstream effector, caspase-1. CONCLUSIONS: These results show that maternal stress during late gestation increases the susceptibility of offspring-particularly males-to the deleterious effects of prenatal air pollutant exposure, which may be due to a synergism of these factors acting on innate immune recognition genes and downstream neuroinflammatory cascades within the developing brain.

FACS analysis of neuronal-glial interactions in the nucleus accumbens following morphine administration.

RATIONALE: Glia, including astrocytes and microglia, can profoundly modulate neuronal function and behavior; however, very little is known about the signaling molecules that govern neuronal-glial communication and in turn affect behavior. Morphine treatment activates microglia and astrocytes in the nucleus accumbens (NAcc) to induce the synthesis of cytokines and chemokines, and this has important implications for addictive behavior. Blocking morphine-induced glial activation using the nonspecific glial inhibitor, ibudilast, has no effect on the initial rewarding properties of morphine, but completely prevents the relapse of drug-seeking behavior months later. OBJECTIVES: We sought to determine the cellular source of these cytokines and chemokines in the NAcc in response to morphine, and the cell-type-specific expression pattern of their receptors to determine whether neurons have the capacity to respond to these immune signals directly. METHODS: We used fluorescence-activated cell sorting of neurons (Thy1+), astrocytes (GLT1+), and microglia (CD11b+) from the NAcc for the analysis of cell type specific gene expression following morphine or saline treatment. RESULTS: The results indicate that microglia and neurons each produce a subset of chemokines in response to morphine and that neurons have the capacity to respond directly to a select group of these chemokines via their receptors. In addition, we provide evidence that microglia are capable of responding directly to dopamine release in the NAcc. CONCLUSIONS: Future studies will examine the mechanism(s) by which neurons respond to these immune signals produced by microglia in an effort to understand their effect on addictive behaviors.

Prenatal air pollution exposure induces neuroinflammation and predisposes offspring to weight gain in adulthood in a sex-specific manner.

Emerging evidence suggests environmental chemical exposures during critical windows of development may contribute to the escalating prevalence of obesity. We tested the hypothesis that prenatal air pollution exposure would predispose the offspring to weight gain in adulthood. Pregnant mice were exposed to filtered air (FA) or diesel exhaust (DE) on embryonic days (E) 9-17. Prenatal DE induced a significant fetal brain cytokine response at E18 (46-390% over FA). As adults, offspring were fed either a low-fat diet (LFD) or high-fat diet (HFD) for 6 wk. Adult DE male offspring weighed 12% more and were 35% less active than FA male offspring at baseline, whereas there were no differences in females. Following HFD, DE males gained weight at the same rate as FA males, whereas DE females gained 340% more weight than FA females. DE-HFD males had 450% higher endpoint insulin levels than FA-HFD males, and all males on HFD showed decreased activity and increased anxiety, whereas females showed no differences. Finally, both DE males and females fed HFD showed increased microglial activation (30-66%) within several brain regions. Thus, prenatal air pollution exposure can "program" offspring for increased susceptibility to diet-induced weight gain and neuroinflammation in adulthood in a sex-specific manner.

Regulatory B10 cells differentiate into antibody-secreting cells after transient IL-10 production in vivo.

Regulatory B cells that are functionally defined by their capacity to express IL-10 (B10 cells) downregulate inflammation and autoimmunity. In studies using well-defined IL-10 reporter mice, this rare B10 cell subset was also found to maintain a capacity for plasma cell differentiation. During a transient period of il10 transcription, the blimp1 and irf4 transcription factors were induced in B10 cells, whereas pax5 and bcl6 were downregulated as a significant fraction of B10 cells completed the genetic and phenotypic program leading to Ab-secreting cell differentiation in vitro and in vivo. B10 cell-derived IgM reacted with both self- and foreign Ags, whereas B10 cells generated Ag-specific IgG in response to immunizations. Moreover, B10 cells represented a significant source of serum IgM and IgG during adoptive-transfer experiments and produced Ag-specific, polyreactive and autoreactive Ab specificities that were consistent with their expression of a diverse AgR repertoire. Thereby, B10 cells limit inflammation and immune responses by the transient production of IL-10, and may facilitate clearance of their eliciting Ags through an inherent capacity to quickly generate polyreactive and/or Ag-specific Abs during humoral immune responses.

A lifespan approach to neuroinflammatory and cognitive disorders: a critical role for glia.

Cognitive decline is a common problem of aging. Whereas multiple neural and glial mechanisms may account for these declines, microglial sensitization and/or dystrophy has emerged as a leading culprit in brain aging and dysfunction. However, glial activation is consistently observed in normal brain aging as well, independent of frank neuroinflammation or functional impairment. Such variability suggests the existence of additional vulnerability factors that can impact neuronal-glial interactions and thus overall brain and cognitive health. The goal of this review is to elucidate our working hypothesis that an individual's risk or resilience to neuroinflammatory disorders and poor cognitive aging may critically depend on their early life experience, which can change immune reactivity within the brain for the remainder of the lifespan. For instance, early-life infection in rats can profoundly disrupt memory function in young adulthood, as well as accelerate age-related cognitive decline, both of which are linked to enduring changes in glial function that occur in response to the initial infection. We discuss these findings within the context of the growing literature on the role of immune molecules and neuroimmune crosstalk in normal brain development. We highlight the intrinsic factors (e.g., chemokines, hormones) that regulate microglial development and their colonization of the embryonic and postnatal brain, and the capacity for disruption or "re-programming" of this crucial process by external events (e.g., stress, infection). An impact on glia, which in turn alters neural development, has the capacity to profoundly impact cognitive and mental health function at all stages of life.

Microglia and memory: modulation by early-life infection.

The proinflammatory cytokine interleukin-1ß (IL-1ß) is critical for normal hippocampus (HP)-dependent cognition, whereas high levels can disrupt memory and are implicated in neurodegeneration. However, the cellular source of IL-1ß during learning has not been shown, and little is known about the risk factors leading to cytokine dysregulation within the HP. We have reported that neonatal bacterial infection in rats leads to marked HP-dependent memory deficits in adulthood. However, deficits are only observed if unmasked by a subsequent immune challenge [lipopolysaccharide (LPS)] around the time of learning. These data implicate a long-term change within the immune system that, upon activation with the "second hit," LPS, acutely impacts the neural processes underlying memory. Indeed, inhibiting brain IL-1ß before the LPS challenge prevents memory impairment in neonatally infected (NI) rats. We aimed to determine the cellular source of IL-1ß during normal learning and thereby lend insight into the mechanism by which this cytokine is enduringly altered by early-life infection. We show for the first time that CD11b(+) enriched cells are the source of IL-1ß during normal HP-dependent learning. CD11b(+) cells from NI rats are functionally sensitized within the adult HP and produce exaggerated IL-1ß ex vivo compared with controls. However, an exaggerated IL-1ß response in vivo requires LPS before learning. Moreover, preventing microglial activation during learning prevents memory impairment in NI rats, even following an LPS challenge. Thus, early-life events can significantly modulate normal learning-dependent cytokine activity within the HP, via a specific, enduring impact on brain microglial function.

Amplified B lymphocyte CD40 signaling drives regulatory B10 cell expansion in mice.

BACKGROUND: Aberrant CD40 ligand (CD154) expression occurs on both T cells and B cells in human lupus patients, which is suggested to enhance B cell CD40 signaling and play a role in disease pathogenesis. Transgenic mice expressing CD154 by their B cells (CD154(TG)) have an expanded spleen B cell pool and produce autoantibodies (autoAbs). CD22 deficient (CD22(-/-)) mice also produce autoAbs, and importantly, their B cells are hyper-proliferative following CD40 stimulation ex vivo. Combining these 2 genetic alterations in CD154(TG)CD22(-/-) mice was thereby predicted to intensify CD40 signaling and autoimmune disease due to autoreactive B cell expansion and/or activation. METHODOLOGY/PRINCIPAL FINDINGS: CD154(TG)CD22(-/-) mice were assessed for their humoral immune responses and for changes in their endogenous lymphocyte subsets. Remarkably, CD154(TG)CD22(-/-) mice were not autoimmune, but instead generated minimal IgG responses against both self and foreign antigens. This paucity in IgG isotype switching occurred despite an expanded spleen B cell pool, higher serum IgM levels, and augmented ex vivo B cell proliferation. Impaired IgG responses in CD154(TG)CD22(-/-) mice were explained by a 16-fold expansion of functional, mature IL-10-competent regulatory spleen B cells (B10 cells: 26.7×10(6)±6 in CD154(TG)CD22(-/-) mice; 1.7×10(6)±0.4 in wild type mice, p<0.01), and an 11-fold expansion of B10 cells combined with their ex vivo-matured progenitors (B10+B10pro cells: 66×10(6)±3 in CD154(TG)CD22(-/-) mice; 6.1×10(6)±2 in wild type mice, p<0.01) that represented 39% of all spleen B cells. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.

B-cell homeostasis requires complementary CD22 and BLyS/BR3 survival signals.

Peripheral B-cell numbers are tightly regulated by homeostatic mechanisms that influence the transitional and mature B-cell compartments and dictate the size and clonotypic diversity of the B-cell repertoire. B-lymphocyte stimulator (BLyS, a trademark of Human Genome Sciences, Inc.) plays a key role in regulating peripheral B-cell homeostasis. CD22 also promotes peripheral B-cell survival through ligand-dependent mechanisms. The B-cell subsets affected by the absence of BLyS and CD22 signals overlap, suggesting that BLyS- and CD22-mediated survival are intertwined. To examine this, the effects of BLyS insufficiency following neutralizing BLyS mAb treatment in mice also treated with CD22 ligand-blocking mAb were examined. Combined targeting of the BLyS and CD22 survival pathways led to significantly greater clearance of recirculating bone marrow, blood, marginal zone and follicular B cells than either treatment alone. Likewise, BLyS blockade further reduced bone marrow, blood and spleen B-cell numbers in CD22(-/-) mice. Notably, BLyS receptor expression and downstream signaling were normal in CD22(-/-) B cells, suggesting that CD22 does not directly alter BLyS responsiveness. CD22 survival signals were likewise intact in the absence of BLyS, as CD22 mAb treatment depleted blood B cells from mice with impaired BLyS receptor 3 (BR3) signaling. Finally, enforced BclxL expression, which rescues BR3 impairment, did not affect B-cell depletion following CD22 mAb treatment. Thus, the current studies support a model whereby CD22 and BLyS promote the survival of overlapping B-cell subsets but contribute to their maintenance through independent and complementary signaling pathways.