Expanding the View of IKK: New Substrates and New Biology

The inhibitor of kappa B kinase (IKK) family consists of IKKα, IKKβ, and the IKK-related kinases TBK1 and IKKε. These kinases are considered master regulators of inflammation and innate immunity via their control of the transcription factors NF-κB, IRF3, and IRF7. Novel phosphorylated substrates have been attributed to these kinases, a subset of which is not directly related to either inflammation or innate immunity. These findings have greatly expanded the perspectives on the biological activities of these kinases. In this review we highlight some of the novel substrates for this kinase family and discuss the biological implications of these phosphorylation events

Myeloid TBK1 signaling contributes to the immune response to influenza

Macrophages provide key elements of the host response to influenza A virus (IAV) infection, including expression of type I IFN and inflammatory cytokines and chemokines. TBK1 (TNF receptor–associated factor family member–associated NF-kB activator–binding kinase 1) contributes to IFN expression and antivira responses in some cell types, but its role in the innate response to IA in vivo is unknown. We hypothesized that macrophage TBK1 contributes to both IFN and non-IFN components of host defense and IAV pathology. We generated myeloid-conditional TBK1 knockout mice and assessed the in vitro and in vivo consequences of IAV infection. Myeloid-specific loss of TBK1 in vivo resulted in less sever host response to IAV, as assessed by decreased mortality, weight loss and hypoxia and less inflammatory changes in BAL fluid relative to wild-type mice despite no differences in viral load. Mice lacking myeloid TBK1 showed less recruitment of CD64 1 SiglecF 2 Ly6C hi inflammatory macrophages, less expression of inflammatory cytokines in the BAL fluid, and less expression of both IFN regulatory factor and NF-kB target genes in the lung. Analysis of sorted alveolar macrophages, inflammatory macrophages, and lung interstitial macrophages revealed that each subpopulation requires TBK1 for distinct components of the response to IAV infection. Our findings define roles for myeloid TBK1 in IAV-induced lung inflammation apart from IFN type I expression and point to myeloid TBK1 as a central and cell type–specific regulator of virus-induced lung damage

Overview: Systemic Inflammatory Response Derived From Lung Injury Caused by SARS-CoV-2 Infection Explains Severe Outcomes in COVID-19

Most SARS-CoV2 infections will not develop into severe COVID-19. However, in some patients, lung infection leads to the activation of alveolar macrophages and lung epithelial cells that will release proinflammatory cytokines. IL-6, TNF, and IL-1β increase expression of cell adhesion molecules (CAMs) and VEGF, thereby increasing permeability of the lung endothelium and reducing barrier protection, allowing viral dissemination and infiltration of neutrophils and inflammatory monocytes. In the blood, these cytokines will stimulate the bone marrow to produce and release immature granulocytes, that return to the lung and further increase inflammation, leading to acute respiratory distress syndrome (ARDS). This lung-systemic loop leads to cytokine storm syndrome (CSS). Concurrently, the acute phase response increases the production of platelets, fibrinogen and other pro-thrombotic factors. Systemic decrease in ACE2 function impacts the Renin-Angiotensin-Kallikrein-Kinin systems (RAS-KKS) increasing clotting. The combination of acute lung injury with RAS-KKS unbalance is herein called COVID-19 Associated Lung Injury (CALI). This conservative two-hit model of systemic inflammation due to the lung injury allows new intervention windows and is more consistent with the current knowledge

Effects of IFN-γ on immune cell kinetics during the resolution of acute lung injury

The immunologic responses that occur early in the acute respiratory distress syndrome (ARDS) elicit immune-mediated damage. The mechanisms underlying the resolution of ARDS, particularly the role of signaling molecules in regulating immune cell kinetics, remain important questions. Th1-mediated responses can contribute to the pathogenesis of acute lung injury (ALI). Interferon-gamma (IFN-γ) orchestrates early inflammatory events, enhancing immune-mediated damage. The current study investigated IFN-γ during resolution in several experimental models of ALI. The absence of IFN-γ resulted in altered kinetics of lymphocyte and macrophage responses, suggesting that IFN-γ present in this microenvironment is influential in ALI resolution. Genetic deficiency of IFN-γ or administering neutralizing IFN-γ antibodies accelerated the pace of resolution. Neutralizing IFN-γ decreased the numbers of interstitial and inflammatory macrophages and increased alveolar macrophage numbers during resolution. Our results underline the complexity of lung injury resolution and provide insight into the effects through which altered IFN-γ concentrations affect immune cell kinetics and the rate of resolution. These findings suggest that therapies that spatially or temporally control IFN-γ signaling may promote ALI resolution. Identifying and elucidating the mechanisms critical to ALI resolution will allow the development of therapeutic approaches to minimize collateral tissue damage without adversely altering the response to injury

MTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation

CD4+ T cells lacking the mTORC1 activator Rheb fail to secrete IFN-g under Th1 polarizing conditions. We hypothesized that this phenotype is due to defects in regulation of the canonical Th1 transcription factor T-bet at the level of protein phosphorylation downstream of mTORC1. To test this hypothesis, we employed targeted mass-spectrometry proteomic analysis-multiple reaction monitoring mass spectrometry. We used this method to detect and quantify predicted phosphopeptides derived from T-bet. By analyzing activated murine wild-type and Rheb-deficient CD4+ T cells, as well as murine CD4+ T cells activated in the presence of rapamycin, a pharmacologic inhibitor of mTORC1, we were able to identify six T-bet phosphorylation sites. Five of these are novel, and four sites are consistently dephosphorylated in both Rheb-deficient CD4+ T cells and T cells treated with rapamycin, suggesting mTORC1 signaling controls their phosphorylation. Alanine mutagenesis of each of the six phosphorylation sites was tested for the ability to impair IFN-g expression. Single phosphorylation site mutants still support induction of IFN-g expression; however, simultaneous mutation of three of the mTORC1-dependent sites results in significantly reduced IFN-g expression. The reduced activity of the triple mutant T-bet is associated with its failure to recruit chromatin remodeling complexes to the Ifng gene promoter. These results establish a novel mechanism by which mTORC1 regulates Th1 differentiation, through control of T-bet phosphorylation

p31 comet acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a "wait anaphase" signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31comet, a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31 comet during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31 comet traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31 comet arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31 comet is required for timely mitotic exit. We propose that p31 comet is an essential component of the machinery that silences the checkpoint during each cell cycle

TBK1 Limits mTORC1 by Promoting Phosphorylation of Raptor Ser877

While best known for its role in the innate immune system, the TANK-binding kinase 1 (TBK1) is now known to play a role in modulating cellular growth and autophagy. One of the major ways that TBK1 accomplishes this task is by modulating the mechanistic Target of Rapamycin (mTOR), a master regulator that when activated promotes cell growth and inhibits autophagy. However, whether TBK1 promotes or inhibits mTOR activity is highly cell type and context dependent. To further understand the mechanism whereby TBK1 regulates mTOR, we tested the hypothesis that TBK1 phosphorylates a key component of the mTOR complex 1 (mTORC1), Raptor. Using kinase assays coupled with mass spectrometry, we mapped the position of the TBK1 dependent phosphorylation sites on Raptor in vitro. Among the sites identified in vitro, we found that TBK1 promotes Raptor Ser877 phosphorylation in cells both basally and in response to pathogen-associated molecules known to induce TBK1 activity. The levels of Raptor Ser877 phosphorylation were inversely correlated with the levels of mTOR activity. Expression of a mutant Raptor that could not be phosphorylated at Ser877 led to an increase in mTORC1 activity. We conclude that TBK1 limits mTORC1 activity by promoting Raptor Ser877 phosphorylation

Hyaluronan fragments induce IFNβ via a novel TLR4-TRIF-TBK1-IRF3- dependent pathway

Background: The extracellular matrix plays a critical role in insuring tissue integrity and water homeostasis. However, breakdown products of the extracellular matrix have emerged as endogenous danger signals, designed to rapidly activate the immune system against a potential pathogen breach. Type I interferons play a critical role in the immune response against viral infections. In the lungs, hylauronan (HA) exists as a high molecular weight, biologically inert extracellular matrix component that is critical for maintaining lung function. When lung tissue is injured, HA is broken down into lower molecular weight fragments that alert the immune system to the breach in tissue integrity by activating innate immune responses. HA fragments are known to induce inflammatory gene expression via TLR-MyD88-dependent pathways. Methods. Primary peritoneal macrophages from C57BL/6 wild type, TLR4 null, TLR3 null, MyD88 null, and TRIF null mice as well as alveolar and peritoneal macrophage cell lines were stimulated with HA fragments and cytokine production was assessed by rt-PCR and ELISA. Western blot analysis for IRF3 was preformed on cell lysates from macrophages stimulate with HA fragments. Results: We demonstrate for the first time that IFNβ is induced in murine macrophages by HA fragments. We also show that HA fragments induce IFNβ using a novel pathway independent of MyD88 but dependent on TLR4 via TRIF and IRF-3. Conclusions: Overall our findings reveal a novel signaling pathway by which hyaluronan can modulate inflammation and demonstrate the ability of hyaluronan fragments to induce the expression of type I interferons in response to tissue injury even in the absence of viral infection. This is independent of the pathway of the TLR2-MyD88 used by these matrix fragments to induce inflammatory chemokines. Thus, LMW HA may be modifying the inflammatory milieu simultaneously via several pathways

Transcriptional analysis of Foxp3+ Tregs and functions of two identified molecules during resolution of ALI

Recovery from acute lung injury (ALI) is an active process. Foxp3+ Tregs contribute to recovery from ALI through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. The current study investigates Treg transcriptional profiles during resolution of ALI in mice. Tregs from either lung or splenic tissue were isolated from uninjured mice or mice recovering from ALI and then examined for differential gene expression between these conditions. In mice with ALI, Tregs isolated from the lungs had hundreds of differentially expressed transcripts compared with those from the spleen, indicating that organ specificity and microenvironment are critical in Treg function. These regulated transcripts suggest which intracellular signaling pathways modulate Treg behavior. Interestingly, several transcripts having no prior recognized function in Tregs were differentially expressed by lung Tregs during resolution. Further investigation into 2 identified transcripts, Mmp12 and Sik1, revealed that Treg-specific expression of each plays a role in Treg-promoted ALI resolution. This study provides potentially novel information describing the signals that may expand resident Tregs, recruit or retain them to the lung during ALI, and modulate their function. The results provide insight into both tissue- and immune microenvironment–specific transcriptional differences through which Tregs direct their effects

Utility of a safety switch to abrogate CD19.CAR T-cell–associated neurotoxicity

Chimeric antigen receptor (CAR)-modified T cells targeting the CD19 antigen are approved to treat relapsed and refractory B-cell malignancies.Despite durable objective responses, most patients experience acute toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Although many cases of ICANS resolve with supportive measures, high-grade ICANS may result in status epilepticus, lasting neurologic deficits, cerebral edema, and death. Therefore, novel approaches for severe ICANS fill an urgent unmet need to enhance the use of adoptive cellular therapy