Jocelyn R. Farmer

Human neuronal cells possess functional cytoplasmic and TLR-mediated innate immune pathways influenced by phosphatidylinositol-3 kinase signaling.

Innate immune pathways are early defense responses important for the immediate control and eventual clearance of many pathogens, where signaling is initiated via pattern recognition receptor (PRR)-mediated events that occur in a ligand- and cell-type specific manner. Within CNS neurons, innate immune pathways are likely crucial to control pathogens that target these essential yet virtually irreplaceable cells. However, relatively little is known about the induction and regulation of neuronal PRR signaling. In this report, we used human neuronal cell lines and primary rat neuronal cultures to examine PRR expression and function. We found that several innate immune receptor ligands, including Sendai virus, the dsRNA mimetic polyinosinic-polycytidylic acid, and LPS all activated differentiation-dependent neuronal innate immune pathways. Functional genetic analyses revealed that IFN regulatory factor 3-mediated pathways that resulted in IFN-β transcriptional upregulation were activated in cultured human neuronal cells by the PRRs TLR3, MDA5, or RIG-I in a ligand-specific manner. Furthermore, genome-wide transcriptional array and targeted genetic and pharmacologic analyses identified PI3K signaling as crucial for the induction of innate immune pathways in neurons. These results indicate that human neuronal cells possess specific and functional PRR pathways essential for the effective induction of innate immune responses, and suggest that neurons can play an active role in defense against neurotropic pathogens.

Neurotropic arboviruses induce interferon regulatory factor 3-mediated neuronal responses that are cytoprotective, interferon independent, and inhibited by Western equine encephalitis virus capsid.

ABSTRACT Cell-intrinsic innate immune responses mediated by the transcription factor interferon regulatory factor 3 (IRF-3) are often vital for early pathogen control, and effective responses in neurons may be crucial to prevent the irreversible loss of these critical central nervous system cells after infection with neurotropic pathogens. To investigate this hypothesis, we used targeted molecular and genetic approaches with cultured neurons to study cell-intrinsic host defense pathways primarily using the neurotropic alphavirus western equine encephalitis virus (WEEV). We found that WEEV activated IRF-3-mediated neuronal innate immune pathways in a replication-dependent manner, and abrogation of IRF-3 function enhanced virus-mediated injury by WEEV and the unrelated flavivirus St. Louis encephalitis virus. Furthermore, IRF-3-dependent neuronal protection from virus-mediated cytopathology occurred independently of autocrine or paracrine type I interferon activity. Despite being partially controlled by IRF-3-dependent signals, WEEV also disrupted antiviral responses by inhibiting pattern recognition receptor pathways. This antagonist activity was mapped to the WEEV capsid gene, which disrupted signal transduction downstream of IRF-3 activation and was independent of capsid-mediated inhibition of host macromolecular synthesis. Overall, these results indicate that innate immune pathways have important cytoprotective activity in neurons and contribute to limiting injury associated with infection by neurotropic arboviruses.

Activation of the Type I Interferon Pathway Is Enhanced in Response to Human Neuronal Differentiation

Despite the crucial role of innate immunity in preventing or controlling pathogen-induced damage in most, if not all, cell types, very little is known about the activity of this essential defense system in central nervous system neurons, especially in humans. In this report we use both an established neuronal cell line model and an embryonic stem cell-based system to examine human neuronal innate immunity and responses to neurotropic alphavirus infection in cultured cells. We demonstrate that neuronal differentiation is associated with increased expression of crucial type I interferon signaling pathway components, including interferon regulatory factor-9 and an interferon receptor heterodimer subunit, which results in enhanced interferon stimulation and subsequent heightened antiviral activity and cytoprotective responses against neurotropic alphaviruses such as western equine encephalitis virus. These results identify important differentiation-dependent changes in innate immune system function that control cell-autonomous neuronal responses. Furthermore, this work demonstrates the utility of human embryonic stem cell-derived cultures as a platform to study the interactions between innate immunity, virus infection, and pathogenesis in central nervous system neurons.

Mechanism-Based Strategies for the Management of Autoimmunity and Immune Dysregulation in Primary Immunodeficiencies

A broad spectrum of autoimmunity is now well described in patients with primary immunodeficiencies (PIDs). Management of autoimmune disease in the background of PID is particularly challenging given the seemingly discordant goals of immune support and immune suppression. Our growing ability to define the molecular underpinnings of immune dysregulation has facilitated novel targeted therapeutics. This review focuses on mechanism-based treatment strategies for the most common autoimmune and inflammatory complications of PID including autoimmune cytopenias, rheumatologic disease, and gastrointestinal disease. We aim to provide guidance regarding the rational use of these agents in the complex PID patient population.

Prevalence and clinical challenges among adults with primary immunodeficiency and recombination-activating gene deficiency

To the Editor: Recombination-activating gene (RAG) deficiency has an estimated disease incidence of 1:181,000, including severe combined immunodeficiency (SCID) at a rate of 1:330,000. Complete or hypomorphic variants of SCID secondary to low recombinase activity (<5%) present early with severe infections and/or clinical signs of systemic inflammation, such as severe dermatitis, colitis, or both. Hypomorphic RAG1/2 mutations with more preserved residual V(D)J recombination activity (5% to 30%) result in a distinct phenotype of combined immunodeficiency with granuloma, autoimmunity, or both. Beyond combined immunodeficiency, RAG deficiency has been found in patients with predominantly primary antibody deficiencies and naive CD4 T-cell lymphopenia in most cases. Currently, there is no published systematic evaluation for the presence of an underlying RAG deficiency in patients with primary antibody deficiencies. There is great variability among diagnostic modalities for evaluation and treatment for inflammatory lung disease in case reports of RAG deficiency with no standardized guidelines. Clinical features and lung disease for patients with late presentation of RAG deficiency have not been studied extensively. In addition, no studies have examined the prevalence of RAG deficiency in cohorts of adults with primary immunodeficiency (PID). Here we describe a cohort of 15 patients with late presentation of RAG deficiency. We also estimate the prevalence of RAG deficiency in adults with PID after genetic analysis in 2 separate large cohorts of patients with PID. We have analyzed the canonical regions of RAG1 and RAG2 in a total of 692 patients with PID from 2 separate cohorts, one from the United Kingdom (UK) and one from Austria (Vienna). The UK cohort is part of the National Institute for Health Research BioResource–Rare Diseases PID study, as previously described (Tuijnenburg et al). In the National Institute for Health Research BioResource–Rare Diseases PID cohort of 558 patients (299 adults) and the Vienna cohort of 134 patients (106 adults), we report a total of 5 newly identified cases of RAG deficiency. For details, see the Methods section and Tables E1 to E3 in this article’s Online Repository at www.jacionline.org. Based on these findings, we estimate that the prevalence of RAG deficiency in adults with PID ranges from 1% to 1.9%. For all adult patients with PID currently registered with the UK Primary Immunodeficiency Network database (3294 patients older than age 18 years), we expect to find an additional 32.9 to 62.6 cases of RAG deficiency. Gene variants are shown in Fig 1, A. Cohort demographics are discussed in the Methods section in this article’s Online Repository. Functional characterization of novel RAG variants is discussed in the Methods section in this article’s Online Repository. The activity of mutant RAG1 and RAG2 proteins normally required for catalyzingV(D)J recombination events are shown in Table E2. In addition to the method previously described, we also used a

Common Variable Immunodeficiency Non-Infectious Disease Endotypes Redefined Using Unbiased Network Clustering in Large Electronic Datasets

Common variable immunodeficiency (CVID) is increasingly recognized for its association with autoimmune and inflammatory complications. Despite recent advances in immunophenotypic and genetic discovery, clinical care of CVID remains limited by our inability to accurately model risk for non-infectious disease development. Herein, we demonstrate the utility of unbiased network clustering as a novel method to analyze inter-relationships between non-infectious disease outcomes in CVID using databases at the United States Immunodeficiency Network (USIDNET), the centralized immunodeficiency registry of the United States, and Partners, a tertiary care network in Boston, MA, USA, with a shared electronic medical record amenable to natural language processing. Immunophenotypes were comparable in terms of native antibody deficiencies, low titer response to pneumococcus, and B cell maturation arrest. However, recorded non-infectious disease outcomes were more substantial in the Partners cohort across the spectrum of lymphoproliferation, cytopenias, autoimmunity, atopy, and malignancy. Using unbiased network clustering to analyze 34 non-infectious disease outcomes in the Partners cohort, we further identified unique patterns of lymphoproliferative (two clusters), autoimmune (two clusters), and atopic (one cluster) disease that were defined as CVID non-infectious endotypes according to discrete and non-overlapping immunophenotypes. Markers were both previously described {high serum IgE in the atopic cluster [odds ratio (OR) 6.5] and low class-switched memory B cells in the total lymphoproliferative cluster (OR 9.2)} and novel [low serum C3 in the total lymphoproliferative cluster (OR 5.1)]. Mortality risk in the Partners cohort was significantly associated with individual non-infectious disease outcomes as well as lymphoproliferative cluster 2, specifically (OR 5.9). In contrast, unbiased network clustering failed to associate known comorbidities in the adult USIDNET cohort. Together, these data suggest that unbiased network clustering can be used in CVID to redefine non-infectious disease inter-relationships; however, applicability may be limited to datasets well annotated through mechanisms such as natural language processing. The lymphoproliferative, autoimmune, and atopic Partners CVID endotypes herein described can be used moving forward to streamline genetic and biomarker discovery and to facilitate early screening and intervention in CVID patients at highest risk for autoimmune and inflammatory progression.

Case 28-2017. A 13-Month-Old Girl with Pneumonia and a 33-Year-Old Woman with Hip Pain.

A 13-month-old girl presented with pneumonia. Imaging studies revealed splenomegaly, splenic lesions, and diffuse lymphadenopathy. Three months later, her mother presented with right hip pain and a lytic lesion in the femoral neck. Diagnoses were made.

Reduced numbers of circulating group 2 innate lymphoid cells in patients with common variable immunodeficiency

Recent studies identified an emerging role of group 2 and 3 innate lymphoid cells (ILCs) as key players in the generation of T‐dependent and T‐independent antibody production. In this retrospective case‐control study, CD117+ ILCs (including the majority of ILC2 and ILC3) were reduced in patients with common variable immunodeficiency (CVID). The reduction in CD117+ ILCs was distinctive to CVID and could not be observed in patients with X‐linked agammaglobulinemia. Patients with a more pronounced reduction in CD117+ ILC numbers showed significantly lower numbers of peripheral MZ‐like B cells and an increased prevalence of chronic, non‐infectious enteropathy. Subsequent phenotyping of ILC subsets in CVID revealed that the reduction in CD117+ ILC numbers is due to a reduction in ILC2 numbers. In vitro expansion of CVID ILC2 in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired. Furthermore, upregulation of MHCII and IL‐2RA in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired in CVID ILC2. Thus, our results indicate a dysregulation of ILC subsets with a reduction in ILC2 numbers in CVID, however, further studies are needed to explore whether ILC abnormalities are a primary finding or secondary to disease complications encountered in CVID.

Bilateral Lung Transplantation in a Patient with Humoral Immune Deficiency: A Case Report with Review of the Literature

Humoral immune deficiencies have been associated with noninfectious disease complications including autoimmune cytopenias and pulmonary disease. Herein we present a patient who underwent splenectomy for autoimmune cytopenias and subsequently was diagnosed with humoral immune deficiency in the context of recurrent infections. Immunoglobulin analysis prior to initiation of intravenous immunoglobulin (IVIG) therapy was notable for low age-matched serum levels of IgA (11 mg/dL), IgG2 (14 mg/L), and IgG4 (5 mg/L) with a preserved total level of IgG. Flow cytometry was remarkable for B cell maturation arrest at the IgM+/IgD+ stage. Selective screening for known primary immune deficiency-causing genetic defects was negative. The disease course was uniquely complicated by the development of pulmonary arteriovenous malformations (AVMs), ultimately requiring bilateral lung transplantation in 2012. This is a patient with humoral immune deficiency that became apparent only after splenectomy, which argues for routine immunologic evaluation prior to vaccination and splenectomy. Lung transplantation is a rare therapeutic endpoint and to our knowledge has never before been described in a patient with humoral immune deficiency for the indication of pulmonary AVMs.

Congenital Immunodeficiency Diseases: Crossroad of Infection, Autoimmunity, and Hyperinflammation

Primary immunodeficiency disorders (PIDs) are genetically predetermined conditions of innate or adaptive immune system dysregulation. Susceptibility toward infection has been a long-standing, often diagnosis-requiring, feature. More recently, autoimmunity as a central pathology in PIDs has been described. Our growing ability to link unique genetic defects with downstream mechanisms that drive hyperinflammation and loss of self-tolerance is extremely powerful. Ultimately, successful management of the PID patient requires not only prevention of infection, but early detection and treatment of autoimmune complications through the use of mechanism-based therapies, which will directly address the immune dysregulation at play.