Stephen R. Brooks

An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome

Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1β and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion led to successful treatment with IL-1–blocking agents. Herein we report a de novo missense mutation (c.1009A>T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1β and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4–transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.

BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb.

Asymmetric Action of STAT Transcription Factors Drives Transcriptional Outputs and Cytokine Specificity

Interleukin-6 (IL-6) and IL-27 signal through a shared receptor subunit and employ the same downstream STAT transcription proteins, but yet are ascribed unique and overlapping functions. To evaluate the specificity and redundancy for these cytokines, we quantified their global transcriptomic changes and determined the relative contributions of STAT1 and STAT3 using genetic models and chromatin immunoprecipitation-sequencing (ChIP-seq) approaches. We found an extensive overlap of the transcriptomes induced by IL-6 and IL-27 and few examples in which the cytokines acted in opposition. Using STAT-deficient cells and T cells from patients with gain-of-function STAT1 mutations, we demonstrated that STAT3 is responsible for the overall transcriptional output driven by both cytokines, whereas STAT1 is the principal driver of specificity. STAT1 cannot compensate in the absence of STAT3 and, in fact, much of STAT1 binding to chromatin is STAT3 dependent. Thus, STAT1 shapes the specific cytokine signature superimposed upon STAT3s action.

Tofacitinib Ameliorates Murine Lupus and Its Associated Vascular Dysfunction

Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized.

Effects of HLA-B27 on Gut Microbiota in Experimental Spondyloarthritis Implicate an Ecological Model of Dysbiosis

To investigate whether HLA–B27–mediated experimental spondyloarthritis (SpA) is associated with a common gut microbial signature, in order to identify potential drivers of pathogenesis.

The Transcription Factor T-bet Limits Amplification of Type I IFN Transcriptome and Circuitry in T Helper 1 Cells

Summary Host defense requires the specification of CD4+ helper T (Th) cells into distinct fates, including Th1 cells that preferentially produce interferon‐&ggr; (IFN‐&ggr;). IFN‐&ggr;, a member of a large family of anti‐pathogenic and anti‐tumor IFNs, induces T‐bet, a lineage‐defining transcription factor for Th1 cells, which in turn supports IFN‐&ggr; production in a feed‐forward manner. Herein, we show that a cell‐intrinsic role of T‐bet influences how T cells perceive their secreted product in the environment. In the absence of T‐bet, IFN‐&ggr; aberrantly induced a type I IFN transcriptomic program. T‐bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, in addition to promoting Th1 effector commitment, T‐bet acts as a repressor in differentiated Th1 cells to prevent abberant autocrine type I IFN and downstream signaling. Graphical Abstract Figure. No Caption available. HighlightsT‐bet‐repressed genes are enriched with type‐I‐IFN‐stimulated genes (ISGs)T‐bet constrains activity of type I IFN transcription factors in IFN‐&ggr; conditionsBlocking IFNAR rectifies aberrant ISG expression by IFN‐&ggr; in T‐bet knockout cellsT‐bet restrains IFN‐&ggr;‐induced collateral type I IFN circuitry in the Th1 response in vivo &NA; T‐bet directs T helper 1 cell differentiation and IFN‐&ggr; production. Iwata et al. find that T‐bet also acts as a repressor of type I IFN transcription factors and type‐I‐IFN‐stimulated genes and collectively restrains IFN‐&ggr;‐induced collateral type I IFN circuitry during the Th1 response.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors

Objectives To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. Methods We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients’ primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). Results We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1β were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients’ fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. Conclusions Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

A157: Macrophage Activation Syndrome‐like Illness Due to an Activating Mutation in NLRC4

Macrophage Activation Syndrome (MAS) is a life‐threatening systemic inflammatory disorder of unknown etiology. While MAS has no known genetic basis, clinical similarity with a genetic disorder of impaired cytotoxicity known as primary Hemophagocytic Lymphohistiocytosis (HLH) has suggested shared pathogenesis. In contrast, other investigations have suggested innate immune dysregulation drives MAS.

Transcriptional signature primes human oral mucosa for rapid wound healing

Transcriptional profiling of human cutaneous and oral wound healing reveals pathways involved in rapid wound resolution. Rapid repair Wounds in the mouth heal faster and with less scarring than wounds in other locations on the body. To understand differences in healing, Iglesias-Bartolome et al. performed transcriptional analysis on sequential, paired oral and skin biopsies from healthy human subjects. Compared to baseline, skin samples showed a larger number of up-regulated genes on subsequent biopsies than oral samples, indicating that healing was unresolved. Oral wounds healed faster than skin wounds, and certain transcription factors were consistently up-regulated in the oral wounds but not in skin wounds. Overexpressing some of these transcription factors in a mouse model of skin wounding enhanced healing. The authors suggest that the molecular signature of the oral mucosa could be used to develop therapies for wound healing. Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound–related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments.

IL-10 induces a STAT3-dependent autoregulatory loop in TH2 cells that promotes Blimp-1 restriction of cell expansion via antagonism of STAT5 target genes

STAT3-mediated Blimp-1 induction in T cells antagonizes STAT5’s role in promoting cell cycle and cell survival genes. Regulating Blimp-1 in TH2 cells The transcription factor Blimp-1 has been reported to limit autoimmunity in T cells. Now, Poholek et al. explore the mechanisms regulating Blimp-1 expression in T helper 2 (TH2) cells. They found that the cytokine interleukin-10 (IL-10) induced Blimp-1 expression in TH2 cells through STAT3. Signal transducer and activator of transcription 3 (STAT3) and Blimp-1 then acted together to boost IL-10 expression in a positive regulatory loop. The induced Blimp-1 then limited cell expansion by inhibiting STAT5 induction of cell cycle and antiapoptotic genes. Thus, by activating Blimp-1, IL-10 may prevent cell expansion, thus restraining autoimmunity. Blimp-1 expression in T cells extinguishes the fate of T follicular helper cells, drives terminal differentiation, and limits autoimmunity. Although various factors have been described to control Blimp-1 expression in T cells, little is known about what regulates Blimp-1 expression in T helper 2 (TH2) cells and the molecular basis of its actions. We report that signal transducer and activator of transcription 3 (STAT3) unexpectedly played a critical role in regulating Blimp-1 in TH2 cells. Furthermore, we found that the cytokine interleukin-10 (IL-10) acted directly on TH2 cells and was necessary and sufficient to induce optimal Blimp-1 expression through STAT3. Together, Blimp-1 and STAT3 amplified IL-10 production in TH2 cells, creating a strong autoregulatory loop that enhanced Blimp-1 expression. Increased Blimp-1 in T cells antagonized STAT5-regulated cell cycle and antiapoptotic genes to limit cell expansion. These data elucidate the signals required for Blimp-1 expression in TH2 cells and reveal an unexpected mechanism of action of IL-10 in T cells, providing insights into the molecular underpinning by which Blimp-1 constrains T cell expansion to limit autoimmunity.