Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-κB signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.

IRF5 is a novel regulator of CXCL13 expression in breast cancer that regulates CXCR5(+) B- and T-cell trafficking to tumor-conditioned media.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration--a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5-positive and -negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5-positive and -negative MDA-MB-231 cells grown in three-dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5-positive tumor-conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme-linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5(+) B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

Cell cycle- and DNA repair pathway-specific effects of apoptosis on tumor suppression.

The DNA damage response comprises DNA repair, cell-cycle checkpoint control, and DNA damage-induced apoptosis that collectively promote genomic integrity and suppress tumorigenesis. Previously, we have shown that the Chk2 kinase functions independently of the Mre11 complex (Mre11, Rad50, and Nbs1) and ATM in apoptosis and suppresses tumorigenesis resulting from hypomorphic alleles of Mre11 or Nbs1. Based on this work, we have proposed that Chk2 limits the oncogenic potential of replication-associated DNA damage. Here we further address the role of Chk2 and damage-induced apoptosis in suppressing the oncogenic potential of chromosome breaks. We show that loss of Chk2 or a mutation in p53 (R172P), which selectively impairs its function in apoptosis, rescued the lethality of mice lacking Lig4, a ligase required for nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks in G0/G1. In contrast to Lig4(-/-)p53(-/-) mice, Lig4(-/-)Chk2(-/-) and Lig4(-/-)p53(R172P/R172P) mice were not prone to organ-specific, rapid tumorigenesis. Although the severe NHEJ deficiency of Lig4(-/-) was a less potent initiator of tumorigenesis in the p53(R172P/R172P) and Chk2(-/-) backgrounds, where p53 cell-cycle functions are largely intact, even mild defects in the intra-S and G2/M checkpoints caused by mutations in Nbs1 are sufficient to influence malignancy in p53(R172P/R172P) mice. We conclude that the oncogenic potential of double-strand breaks resulting from NHEJ deficiency is highly restricted by nonapoptotic functions of p53, such as the G1/S checkpoint or senescence, suggesting that the particular facets of the DNA damage response required for tumor suppression are dictated by the proliferative status of the tumor-initiating cell.

Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as) phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway.

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.

B cell transcription factors: Potential new therapeutic targets for SLE.

B cells represent a critical arm of the adaptive immune system, and under normal circumstances, provide long lasting immunity to a wide range of pathogens. To achieve this, B cells differentiate into specialized subpopulations, which perform unique functions in response to antigen. A complex network of transcription factors regulates this transition as well as subsequent effector functions. Dysregulation of these transcription factors can lead to altered B cell biology and potentially autoimmunity. Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by increased autoantibody production and B cell hyper-reactivity. It still remains unclear, however, what factors contribute to disease pathogenesis. Recent work has suggested dysregulation of B cell transcription factors may play a role in disease onset. Given the prominent role B cells play in the pathogenesis of SLE, it is important to have a full understanding of what transcription factors regulate B cell development, and how dysregulation of some of these transcription factors may contribute to the occurrence and/or pathogenesis of SLE. A comprehensive review of where this field currently stands, what new potential targets have recently been identified, and what functional information is still missing will be discussed.

Distinct fibroblast functions associated with fibrotic and immune-mediated inflammatory diseases and their implications for therapeutic development.

Fibroblasts are ubiquitous cells that can adopt many functional states. As tissue-resident sentinels, they respond to acute damage signals and shape the earliest events in fibrotic and immune-mediated inflammatory diseases. Upon sensing an insult, fibroblasts produce chemokines and growth factors to organize and support the response. Depending on the size and composition of the resulting infiltrate, these activated fibroblasts may also begin to contract or relax thus changing local stiffness within the tissue. These early events likely contribute to the divergent clinical manifestations of fibrotic and immune-mediated inflammatory diseases. Further, distinct changes to the cellular composition and signaling dialogue in these diseases drive progressive fibroblasts specialization. In fibrotic diseases, fibroblasts support the survival, activation and differentiation of myeloid cells, granulocytes and innate lymphocytes, and produce most of the pathogenic extracellular matrix proteins. Whereas, in immune-mediated inflammatory diseases, sequential accumulation of dendritic cells, T cells and B cells programs fibroblasts to support local, destructive adaptive immune responses. Fibroblast specialization has clear implications for the development of effective induction and maintenance therapies for patients with these clinically distinct diseases.

Frontline Science: AMPK regulates metabolic reprogramming necessary for interferon production in human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play a crucial role in innate viral immunity as the most potent producers of type I interferons (IFN) in the human body. However, the metabolic regulation of IFN production in such vast quantity remains poorly understood. In this study, AMP-activated protein kinase (AMPK) is strongly implicated as a driver of metabolic reprogramming that the authors and others have observed in pDCs after activation via TLR7/9. Oxygen consumption and mitochondrial membrane potential (MMP) were elevated following stimulation of pDCs with influenza or herpes simplex virus. Blocking these changes using mitochondrial inhibitors abrogated IFN-α production. While it appears that multiple carbon sources can be used by pDCs, blocking pyruvate metabolism had the strongest effect on IFN-α production. Furthermore, we saw no evidence of aerobic glycolysis (AG) during pDC activation and blocking lactate dehydrogenase activity did not inhibit IFN-α. TLR7/9 ligation induces a posttranslational modification in Raptor that is catalyzed by AMPK, and blocking TLR7/9 before virus introduction prevents this change. Finally, it is demonstrated that Dorsomorphin, an AMPK inhibitor, inhibited both IFN-α production and MMP in a dose-dependent manner. Taken together, these data reveal a potential cellular mechanism for the metabolic reprogramming in TLR 7/9-activated pDCs that supports activation and IFN-α production.

The Interleukin-1 Receptor-Associated Kinase 4 Inhibitor PF-06650833 Blocks Inflammation in Preclinical Models of Rheumatic Disease and in Humans Enrolled in a Randomized Clinical Trial.

OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Inhibition of IRF5 hyperactivation protects from lupus onset and severity.

The transcription factor IFN regulatory factor 5 (IRF5) is a central mediator of innate and adaptive immunity. Genetic variations within IRF5 are associated with a risk of systemic lupus erythematosus (SLE), and mice lacking Irf5 are protected from lupus onset and severity, but how IRF5 functions in the context of SLE disease progression remains unclear. Using the NZB/W F1 model of murine lupus, we show that murine IRF5 becomes hyperactivated before clinical onset. In patients with SLE, IRF5 hyperactivation correlated with dsDNA titers. To test whether IRF5 hyperactivation is a targetable function, we developed inhibitors that are cell permeable, nontoxic, and selectively bind to the inactive IRF5 monomer. Preclinical treatment of NZB/W F1 mice with an inhibitor attenuated lupus pathology by reducing serum antinuclear autoantibodies, dsDNA titers, and the number of circulating plasma cells, which alleviated kidney pathology and improved survival. Clinical treatment of MRL/lpr and pristane-induced lupus mice with an inhibitor led to significant reductions in dsDNA levels and improved survival. In ex vivo human studies, the inhibitor blocked SLE serum-induced IRF5 activation and reversed basal IRF5 hyperactivation in SLE immune cells. We believe this study provides the first in vivo clinical support for treating patients with SLE with an IRF5 inhibitor.

RNAi Transfection Optimized in Primary Naïve B Cells for the Targeted Analysis of Human Plasma Cell Differentiation.

Upon antigen recognition, naïve B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs), called plasma cells. Increased circulating plasma cells are reported in patients with B cell-associated malignancies, chronic graft-vs.-host disease, and autoimmune disorders. Our aim was to optimize an RNAi-based method that efficiently and reproducibly knocks-down genes of interest in human primary peripheral B cells for the targeted analysis of ASC differentiation. The unique contributions of transcriptional diversity in species-specific regulatory networks and the mechanisms of gene function need to be approached directly in human B cells with tools to hone our basic inferences from animal models to human biology. To date, methods for gene knockdown in human primary B cells, which tend to be more refractory to transfection than immortalized B cell lines, have been limited by losses in cell viability and ineffective penetrance. Our single-step siRNA nucleofector-based approach for human primary naïve B cells demonstrates reproducible knockdown efficiency (~40-60%). We focused on genes already known to play key roles in murine ASC differentiation, such as interferon regulatory factor 4 (IRF4) and AID. This study reports a validated non-viral method of siRNA delivery into human primary B cells that can be applied to study gene regulatory networks that control human ASC differentiation.

B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation.

Upon recognition of antigen, B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs). During this transition, B cells are reliant upon a multilayer transcription factor network to achieve a dramatic remodeling of the B cell transcriptional landscape. Increased levels of ASCs are often seen in autoimmune diseases and it is believed that altered expression of regulatory transcription factors play a role in this imbalance. The transcription factor interferon regulatory factor 5 (IRF5) is one such candidate as polymorphisms in IRF5 associate with risk of numerous autoimmune diseases and correlate with elevated IRF5 expression. IRF5 genetic risk has been widely replicated in systemic lupus erythematosus (SLE), and loss of Irf5 ameliorates disease in murine lupus models, in part, through the lack of pathogenic autoantibody secretion. It remains unclear, however, whether IRF5 is contributing to autoantibody production through a B cell-intrinsic function. To date, IRF5 function in healthy human B cells has not been characterized. Using human primary naive B cells, we define a critical intrinsic role for IRF5 in B cell activation, proliferation, and plasmablast differentiation. Targeted IRF5 knockdown resulted in significant immunoglobulin (Ig) D retention, reduced proliferation, plasmablast differentiation, and IgG secretion. The observed decreases were due to impaired B cell activation and clonal expansion. Distinct from murine studies, we identify and confirm new IRF5 target genes, IRF4, ERK1, and MYC, and pathways that mediate IRF5 B cell-intrinsic function. Together, these results identify IRF5 as an early regulator of human B cell activation and provide the first dataset in human primary B cells to map IRF5 dysfunction in SLE.

Specific detection of interferon regulatory factor 5 (IRF5): A case of antibody inequality.

Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors. IRF5 was first identified and characterized as a transcriptional regulator of type I interferon expression after virus infection. In addition to its critical role(s) in the regulation and development of host immunity, subsequent studies revealed important roles for IRF5 in autoimmunity, cancer, obesity, pain, cardiovascular disease, and metabolism. Based on these important disease-related findings, a large number of commercial antibodies have become available to study the expression and function of IRF5. Here we validate a number of these antibodies for the detection of IRF5 by immunoblot, flow cytometry, and immunofluorescence or immunohistochemistry using well-established positive and negative controls. Somewhat surprising, the majority of commercial antibodies tested were unable to specifically recognize human or mouse IRF5. We present data on antibodies that do specifically recognize human or mouse IRF5 in a particular application. These findings reiterate the importance of proper controls and molecular weight standards for the analysis of protein expression. Given that dysregulated IRF5 expression has been implicated in the pathogenesis of numerous diseases, including autoimmune and cancer, results indicate that caution should be used in the evaluation and interpretation of IRF5 expression analysis.

Rad50 is dispensable for the maintenance and viability of postmitotic tissues.

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.