Halli E. Miller

Inflammatory Signals Direct Expression of Human IL12RB1 into Multiple Distinct Isoforms

IL12RB1 is essential for human resistance to multiple intracellular pathogens, including Mycobacterium tuberculosis. In its absence, the proinflammatory effects of the extracellular cytokines IL-12 and IL-23 fail to occur, and intracellular bacterial growth goes unchecked. Given the recent observation that mouse leukocytes express more than one isoform from il12rb1, we examined whether primary human leukocytes similarly express more than one isoform from IL12RB1. We observed that human leukocytes express as many as 13 distinct isoforms, the relative levels of each being driven by inflammatory stimuli both in vitro and in vivo. Surprisingly, the most abundant isoform present before stimulation is a heretofore uncharacterized intracellular form of the IL-12R (termed “isoform 2”) that presumably has limited contact with extracellular cytokine. After stimulation, primary PBMCs, including the CD4+, CD8+, and CD56+ lineages contained therein, alter the splicing of IL12RB1 RNA to increase the relative abundance of isoform 1, which confers IL-12/IL-23 responsiveness. These data demonstrate both a posttranscriptional mechanism by which cells regulate their IL-12/IL-23 responsiveness, and that leukocytes primarily express IL12RB1 in an intracellular form located away from extracellular cytokine.

Early control of Mycobacterium tuberculosis infection requires il12rb1 expression by rag1-dependent lineages.

ABSTRACT IL12RB1 is essential for human resistance to Mycobacterium tuberculosis infection. In the absence of a functional IL12RB1 allele, individuals exhibit susceptibility to disseminated, recurrent mycobacterial infections that are associated with defects in both RAG1-dependent and RAG1-independent hematopoietic lineages. Despite this well-established association, a causal relationship between M. tuberculosis susceptibility and IL12RB1 deficiency in either RAG1-dependent or RAG1-independent lineages has never been formally tested. Here, we use the low-dose aerosol model of experimental tuberculosis (TB) to both establish that infected il12rb1 −/− mice recapitulate important aspects of TB in IL12RB1 null individuals and, more importantly, use radiation bone marrow chimeras to demonstrate that restriction of il12rb1 deficiency solely to rag1-dependent lineages (i.e., T and B cells) allows for the full transfer of the il12rb1 −/− phenotype. We further demonstrate that the protection afforded by adaptive lymphocyte il12rb1 expression is mediated partially through ifng and that, within the same infection, il12rb1-sufficient T cells exhibit dominance over il12rb1-deficient T cells by enhancing ifng expression in the latter population. Collectively, our data establish a basic framework in which to understand how IL12RB1 promotes control of this significant human disease.

Immune-checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T cell activation when expressed on antigen-presenting cells. Vsir−/− mice developed loss of peripheral tolerance and multi-organ chronic inflammatory phenotypes. Vsir−/− CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether or not VISTA regulates innate immunity is unknown. Using a murine model of psoriasis induced by TLR7 agonist imiquimod (IMQ), we show that VISTA deficiency exacerbated psoriasiform inflammation. Enhanced TLR7 signaling in Vsir−/− dendritic cells (DCs) led to the hyper-activation of Erk1/2 and Jnk1/2, and augmented the production of IL-23. IL-23, in turn, promoted the expression of IL-17A in both TCRγδ+ T cells and CD4+ Th17 cells. Furthermore, VISTA regulates the peripheral homeostasis of CD27− γδ T cells and their activation upon TCR-mediated or cytokine-mediated stimulation. IL-17A-producing CD27− γδ T cells were expanded in the Vsir−/− mice and amplified the inflammatory cascade. In conclusion, this study has demonstrated that VISTA critically regulates the inflammatory responses mediated by DCs and IL-17-producing TCRγδ+ and CD4+ Th17 T cells following TLR7 stimulation. Our finding provides a rationale for therapeutically enhancing VISTA-mediated pathways to benefit the treatment of autoimmune and inflammatory disorders.

The introduction of RNA-DNA differences underlies interindividual variation in the human IL12RB1 mRNA repertoire

Significance The gene interleukin-12 receptor β1 (IL12RB1) regulates susceptibility to several human diseases, including mycobacterial disease (e.g., tuberculosis). Here, we demonstrate that many of the mRNAs transcribed from IL12RB1 in primary immune cells contain RNA-DNA differences (RDDs). RDDs are nucleotide differences between RNA and its encoding DNA and are introduced posttranscriptionally; in the case of IL12RB1, RDDs are concentrated in cytokine-binding regions that are important for IL12RB1 function. This observation is significant, as it is the first demonstration to our knowledge that a mechanism of sequence diversification exists for a human cytokine receptor. Given IL12RB1’s importance to mycobacterial disease resistance, our data raise the intriguing possibility that individual differences in IL12RB1 RDD introduction contribute to differences in mycobacterial disease susceptibility. Human interleukin 12 and interleukin 23 (IL12/23) influence susceptibility or resistance to multiple diseases. However, the reasons underlying individual differences in IL12/23 sensitivity remain poorly understood. Here we report that in human peripheral blood mononuclear cells (PBMCs) and inflamed lungs, the majority of interleukin-12 receptor β1 (IL12RB1) mRNAs contain a number of RNA-DNA differences (RDDs) that concentrate in sequences essential to IL12Rβ1’s binding of IL12p40, the protein subunit common to both IL-12 and IL-23. IL12RB1 RDDs comprise multiple RDD types and are detectable by next-generation sequencing and classic Sanger sequencing. As a consequence of these RDDs, the resulting IL12Rβ1 proteins have an altered amino acid sequence that could not be predicted on the basis of genomic DNA sequencing alone. Importantly, the introduction of RDDs into IL12RB1 mRNAs negatively regulates IL12Rβ1’s binding of IL12p40 and is sensitive to activation. Collectively, these results suggest that the introduction of RDDs into an individual’s IL12RB1 mRNA repertoire is a novel determinant of IL12/23 sensitivity.

IL12Rβ1ΔTM Is a Secreted Product of il12rb1 That Promotes Control of Extrapulmonary Tuberculosis

ABSTRACT IL12RB1 is a human gene that is important for resistance to Mycobacterium tuberculosis infection. IL12RB1 is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12Rβ1) that associates with IL12p40 and promotes the development of host-protective TH1cells. Recently, we observed that il12rb1—the mouse homolog of IL12RB1—is alternatively spliced by leukocytes to produce a second isoform (IL12Rβ1ΔTM) that has biological properties distinct from IL12Rβ1. Although the expression of IL12Rβ1ΔTM is elicited by M. tuberculosis in vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12Rβ1ΔTM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12Rβ1ΔTM represents a secreted product of il12rb1 that, when absent from mice, compromises their ability to control M. tuberculosis infection in extrapulmonary organs. Furthermore, elevated M. tuberculosis burdens in IL12Rβ1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in TH1 development. Collectively, these data support a model wherein IL12Rβ1ΔTM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating TH cells response to IL-12.

IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis

IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CD11c(hi) cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11c(hi) lineages are absent.

Author Correction: Immune-checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis

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Abstract 2996: Immune checkpoint protein VISTA suppresses Toll-like receptor signaling and the production of inflammatory cytokines

Introduction: V-domain Ig suppressor of T-cell activation (VISTA, gene Vsir) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T cell activation. Previous studies have shown that Vsir-/- mice developed chronic inflammatory phenotypes, and Vsir-/- CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether VISTA regulates innate immunity is still unknown. Methods: Peritoneal macrophages from WT or Vsir-/- mice were isolated and stimulated with TLR agonists such as CpG (TLR9), R848 (TLR7), LPS (TLR4), Pam3csk4 (TLR2), and poly (I:C) (TLR3). Alternatively, human monocyte THP-1 cells overexpressing VISTA were stimulated by TLR2 agonist Pam3CSK4. The activation of TLR signaling pathways and the production of inflammatory cytokines were examined by Western Blotting, gel shift assay, or ELISA. The ubiquitination status of key signaling molecules such as TRAF6, IRAK1/4 and MyD88 was examined by immunoprecipitation and Western Blotting. To examine the role of VISTA in regulating TLR-mediated inflammatory responses in the context of cancer vaccine, tumor-bearing mice were treated with VISTA-specific monoclonal antibody (mAb) and a peptide vaccine containing TLR agonists. The production of inflammatory cytokines and chemokines within the tumor microenvironment (TME) was examined via quantitative RT-PCR. Results: Vsir-/- macrophages were hyper-responsive towards TLR2/4/7/9 agonists, but not TLR3 agonist, resulting in increased production of inflammatory cytokines IL-6, IL-12, and TNFa. Analysis of signaling cascade revealed that VISTA inhibited the activation of MyD88-dependent TLR signaling, via suppressing the activation of MAPKs, and the activation of transcription factors AP-1 and NF-kB. Consistent with the role of VISTA in regulating TLR-mediated innate immunity, treatment with VISTA-blocking mAb augmented levels of inflammatory cytokines and chemokines within the TME, and synergized with TLR/peptide vaccine, resulting in an optimal therapeutic outcome. Conclusions: Our study establishes that VISTA critically regulates the inflammatory responses of myeloid cells mediated by TLR signaling. In the context of cancer vaccine therapy, VISTA-blocking mAb treatment enhanced levels of inflammatory cytokine and chemokines within the TME, which is critical for the development of optimal tumor-specific T cell responses and the tumor-controlling therapeutic outcome. Citation Format: Wenwen Xu, Ying Yuan, Na Li, Yongwei Zheng, Kamal Rajasekaran, Halli Miller, Michael Olson, Demin Wang, Subramaniam Malarkannan, Li Wang. Immune checkpoint protein VISTA suppresses Toll-like receptor signaling and the production of inflammatory cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2017-2996

Characterization of Respirable Aerosols Generated during Routine Laboratory Procedures

Biosafety Level 2 (BSL-2) and Biosafety Level 3 (BSL-3) facility lab personnel routinely perform procedures that are capable of producing respirable aerosols. While these procedures are considered safe when performed in the closed environment of a biosafety cabinet (BSC), there are, nevertheless, limited data regarding the nature of the aerosols these procedures produce. This lack of aerosol data poses a significant challenge to biosafety professionals, who are charged with assessing the risks associated with handling infectious materials and communicating the importance of proper engineering controls to BSL-2/BSL-3 facility staff. This article characterizes the extent and nature of respirable aerosols produced during routine laboratory procedures when these procedures are performed in an open environment (i.e., outside a BSC). As demonstrated, homogenization, vortexing, and pipetting each produce aerosols of differing characteristics and aerogenic potentials. These characteristics have led to the development of an Exposure Risk Model for routine lab methods to assist biosafety professionals in their efforts to prevent inhalation exposures.