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Immune surveillance by natural killer (NK) cells and their recruitment to sites of inflammation renders them susceptible to viral infection, potentially modulating their effector function. Here, we analyzed innate RNA receptor signaling in NK cells downstream of direct Influenza A virus (IAV) infection and its impact on NK cell effector function. Infection of NK cells with IAV resulted in the activation of TBK1, NF-ϰB and subsequent type-I IFN secretion. CRISPR-generated knockouts in primary human NK cells revealed that this effect depended on the antiviral cytosolic RNA receptor RIG-I. Transfection of NK cells with synthetic 3p-dsRNA, a strong RIG-I agonist that mimics viral RNA, resulted in a similar phenotype and rendered NK cells resistant to subsequent IAV infection. Strikingly, both IAV infection and 3p-dsRNA transfection enhanced degranulation and cytokine production by NK cells when exposed to target cells. Thus, RIG-I activation in NK cells both supports their cell intrinsic viral defense and enhances their cytotoxic effector function against target cells. [ABSTRACT FROM AUTHOR]

TBK1 and IKKЄ are related, crucial kinases in antiviral immune signaling pathways downstream of cytosolic nucleic acid receptors such as cGAS and RIG-I-like receptors. Upon activation, they phosphorylate the transcription factors IRF3 and IRF7 and thereby initiate the expression of type I interferons and antiviral effectors. While point mutation-induced loss of TBK1 kinase activity results in clinical hyper-susceptibility to viral infections, a complete lack of TBK1 expression in humans is unexpectedly not associated with diminished antiviral responses. Here, we provide a mechanistic explanation for these so-far unexplained observations by showing that TBK1 controls the protein expression of its related kinase IKKЄ in human myeloid cells. Mechanistically, TBK1 constitutively diminishes the protein stability of IKKЄ independent of TBK1 kinase activity but dependent on its interaction with the scaffold protein TANK. In consequence, depletion of TBK1 protein but not mutation-induced kinase deficiency induces the upregulation of IKKЄ. Due to the functional redundancy of the kinases in cGAS-STING and RIG-I-like receptor signaling in human myeloid cells, enhanced IKKЄ expression can compensate for the loss of TBK1. We show that IKKЄ upregulation is crucial to ensure unmitigated type I interferon production in conditions of TBK1 deficiency: While the type I interferon response to Listeria monocytogenes infection is maintained upon TBK1 loss, it is strongly diminished in cells harboring a kinase-deficient TBK1 variant, in which IKKЄ is not upregulated. Many pathogens induce TBK1 degradation, suggesting that loss of TBK1-mediated destabilization of IKKЄ is a critical backup mechanism to prevent diminished interferon responses upon TBK1 depletion. [ABSTRACT FROM AUTHOR]

CARD8 senses pathogen-associated protease activity and assembles a pyroptosis-inducing inflammasome, but detailed regulatory mechanisms have remained elusive. In this issue of Immunity , Sharif et al. use cryo-EM and biochemical assays to unveil how DPP9 sequesters the inflammasome-forming C-terminal fragment of CARD8 to suppress its activation. [ABSTRACT FROM AUTHOR]

The magnetic properties of paramagnetic species with spin S ＞ 1/2 are parameterized by the familiar g tensor as well as “zero-field splitting” (ZFS) terms that break the degeneracy between spin states even in the absence of a magnetic field. In this work, we determine the mean values and distributions of the ZFS parameters D and E for six Gd(iii) complexes (S = 7/2) and critically discuss the accuracy of such determination. EPR spectra of the Gd(iii) complexes were recorded in glassy frozen solutions at 10 K or below at Q-band (∼34 GHz), W-band (∼94 GHz) and G-band (240 GHz) frequencies, and simulated with two widely used models for the form of the distributions of the ZFS parameters D and E. We find that the form of the distribution of the ZFS parameter D is bimodal, consisting roughly of two Gaussians centered at D and −D with unequal amplitudes. The extracted values of D (σD) for the six complexes are, in MHz: Gd-NO3Pic, 485 ± 20 (155 ± 37); Gd-DOTA/Gd-maleimide-DOTA, −714 ± 43 (328 ± 99); iodo-(Gd-PyMTA)/MOMethynyl-(Gd-PyMTA), 1213 ± 60 (418 ± 141); Gd-TAHA, 1361 ± 69 (457 ± 178); iodo-Gd-PCTA-[12], 1861 ± 135 (467 ± 292); and Gd-PyDTTA, 1830 ± 105 (390 ± 242). The sign of D was adjusted based on the Gaussian component with larger amplitude. We relate the extracted P(D) distributions to the structure of the individual Gd(iii) complexes by fitting them to a model that superposes the contribution to the D tensor from each coordinating atom of the ligand. Using this model, we predict D, σD, and E values for several additional Gd(iii) complexes that were not measured in this work. The results of this paper may be useful as benchmarks for the verification of quantum chemical calculations of ZFS parameters, and point the way to designing Gd(iii) complexes for particular applications and estimating their magnetic properties a priori. [ABSTRACT FROM AUTHOR]

The four Mn(ii) complexes Mn-DOTA, Mn-TAHA, Mn-PyMTA, and Mn-NO3Py were characterized by electron paramagnetic resonance (EPR), electron–nuclear double resonance (ENDOR), and relaxation measurements, to predict their relative performance in the EPR pulse dipolar spectroscopy (PDS) experiments. High spin density localization on the metal ions was proven by ENDOR on 1H, D, 14N, and 55Mn nuclei. The transverse relaxation of the Mn(ii) complexes appears to be slow enough for PDS-based spin–spin distance determination. Rather advantageous ratios of T1/Tm were measured allowing for good relaxation induced dipolar modulation enhancement (RIDME) performance and, in general, fast shot repetitions in any PDS experiment. Relaxation properties of the Mn(ii) complexes correlate with the strengths of their zero field splitting (ZFS). Further, a comparison of Mn(ii)-DOTA and Gd(iii)-DOTA based spin labels is presented. The RIDME technique to measure nanometer-range Mn(ii)–Mn(ii) distances in biomolecules is discussed as an alternative to the well-known DEER technique that often appears challenging in cases of metal–metal distance measurements. The use of a modified kernel function that includes dipolar harmonic overtones allows model-free computation of the Mn(ii)–Mn(ii) distance distributions. Mn(ii)–Mn(ii) distances are computed from RIDME data of Mn-rulers consisting of two Mn-PyMTA complexes connected by a rodlike spacer of defined length. Level crossing effects seem to have only a weak influence on the distance distributions computed from this set of Mn(ii)–Mn(ii) RIDME data. [ABSTRACT FROM AUTHOR]

The article examines the association of vitamin D therapy with decreased cardiovascular-related morbidity and mortality, by modifying cardiac structure and function in patients with chromic kidney disease. Results revealed a reduction in parathyroid hormone levels in patients treated with paricalcitol within four weeks and maintained levels within the normal range throughout the duration of the study. The researchers observed no differences in Doppler measures of diastolic function, including peak early diastolic lateral mitral annular tissue velocity. They observed increased frequency of episodes of hypercalcemia in patients who were administered with paricalcitol compared with those who received placebo.