A pan-cancer analysis implicates human NKIRAS1 as a tumor-suppressor gene.

The NF-κB family of transcription factors and the Ras family of small GTPases are important mediators of proproliferative signaling that drives tumorigenesis and carcinogenesis. The κB-Ras proteins were previously shown to inhibit both NF-κB and Ras activation through independent mechanisms, implicating them as tumor suppressors with potentially broad relevance to human cancers. In this study, we have used two mouse models to establish the relevance of the κB-Ras proteins for tumorigenesis. Additionally, we have utilized a pan-cancer bioinformatics analysis to explore the role of the κB-Ras proteins in human cancers. Surprisingly, we find that the genes encoding κB-Ras 1 (NKIRAS1) and κB-Ras 2 (NKIRAS2) are rarely down-regulated in tumor samples with oncogenic Ras mutations. Reduced expression of human NKIRAS1 alone is associated with worse prognosis in at least four cancer types and linked to a network of genes implicated in tumorigenesis. Our findings provide direct evidence that loss of NKIRAS1 in human tumors that do not carry oncogenic RAS mutations is associated with worse clinical outcomes.

Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae.

This review provides a summary of the recently ratified changes to genus and species nomenclature within the virus family Flaviviridae along with reasons for these changes. First, it was considered that the vernacular terms "flaviviral", "flavivirus", and "flaviviruses" could under certain circumstances be ambiguous due to the same word stem "flavi" in the taxon names Flaviviridae and Flavivirus; these terms could either have referred to all viruses classified in the family Flaviviridae or only to viruses classified in the included genus Flavivirus. To remove this ambiguity, the genus name Flavivirus was changed to Orthoflavivirus by the International Committee on Taxonomy of Viruses (ICTV). Second, all species names in the family were changed to adhere to a newly ICTV-mandated binomial format (e.g., Orthoflavivirus zikaense, Hepacivirus hominis) similar to nomenclature conventions used for species elsewhere in biology. It is important to note, however, that virus names remain unchanged. Here we outline the revised taxonomy of the family Flaviviridae as approved by the ICTV in April 2023.

PDK1 Is Required for Maintenance of CD4+ Foxp3+ Regulatory T Cell Function.

Regulatory T (Treg) cells have an essential role in maintaining immune homeostasis, in part by suppressing effector T cell functions. Phosphoinositide-dependent kinase 1 (PDK1) is a pleiotropic kinase that acts as a key effector downstream of PI3K in many cell types. In T cells, PDK1 has been shown to be critical for activation of NF-κB and AKT signaling upon TCR ligation and is therefore essential for effector T cell activation, proliferation, and cytokine production. Using Treg cell-specific conditional deletion, we now demonstrate that PDK1 is also essential for Treg cell suppressive activity in vivo. Ablation of Pdk1 specifically in Treg cells led to systemic, lethal, scurfy-like inflammation in mice. Genome-wide analysis confirmed that PDK1 is essential for the regulation of key Treg cell signature gene expression and, further, suggested that PDK1 acts primarily to control Treg cell gene expression through regulation of the canonical NF-κB pathway. Consistent with these results, the scurfy-like phenotype of mice lacking PDK1 in Treg cells was rescued by enforced activation of NF-κB downstream of PDK1. Therefore, PDK1-mediated activation of the NF-κB signaling pathway is essential for regulation of Treg cell signature gene expression and suppressor function.

The T1D-associated lncRNA Lnc13 modulates human pancreatic ß cell inflammation by allele-specific stabilization of STAT1 mRNA.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13 Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic ß-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in ß-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in ß-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic ß-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.

History and classification of Aigai virus (formerly Crimean-Congo haemorrhagic fever virus genotype VI).

Crimean-Congo haemorrhagic fever virus (CCHFV) is the medically most important member of the rapidly expanding bunyaviral family Nairoviridae. Traditionally, CCHFV isolates have been assigned to six distinct genotypes. Here, the International Committee on Taxonomy of Viruses (ICTV) Nairoviridae Study Group outlines the reasons for the recent decision to re-classify genogroup VI (aka Europe-2 or AP-92-like) as a distinct virus, Aigai virus (AIGV).

Guidance for creating individual and batch latinized binomial virus species names.

The International Committee on Taxonomy of Viruses recently adopted, and is gradually implementing, a binomial naming format for virus species. Although full Latinization of these names remains optional, a standardized nomenclature based on Latinized binomials has the advantage of comparability with all other biological taxonomies. As a language without living native speakers, Latin is more culturally neutral than many contemporary languages, and words built from Latin roots are already widely used in the language of science across the world. Conversion of established species names to Latinized binomials or creation of Latinized binomials de novo may seem daunting, but the rules for name creation are straightforward and can be implemented in a formulaic manner. Here, we describe approaches, strategies and steps for creating Latinized binomials for virus species without prior knowledge of Latin. We also discuss a novel approach to the automated generation of large batches of novel genus and species names. Importantly, conversion to a binomial format does not affect virus names, many of which are created from local languages.

Jingchuvirales: a New Taxonomical Framework for a Rapidly Expanding Order of Unusual Monjiviricete Viruses Broadly Distributed among Arthropod Subphyla.

Technical advances in metagenomics and metatranscriptomics have dramatically accelerated virus discovery in recent years. "Chuviruses" were first described in 2015 as obscure negative-sense RNA viruses of diverse arthropods. Although "chuviruses" first appeared to be members of the negarnaviricot order Mononegavirales in phylogenetic analyses using RNA-directed RNA polymerase sequences, further characterization revealed unusual gene orders in genomes that are nonsegmented, segmented, and/or possibly circular. Consequently, a separate order, Jingchuvirales, was established to include a monospecific family, Chuviridae. Recently, it has become apparent that jingchuvirals are broadly distributed and are therefore likely of ecological and economic importance. Here, we describe recent and ongoing efforts to create the necessary taxonomic framework to accommodate the expected flood of novel viruses belonging to the order.

L-RAPiT: A Cloud-Based Computing Pipeline for the Analysis of Long-Read RNA Sequencing Data.

Long-read sequencing (LRS) has been adopted to meet a wide variety of research needs, ranging from the construction of novel transcriptome annotations to the rapid identification of emerging virus variants. Amongst other advantages, LRS preserves more information about RNA at the transcript level than conventional high-throughput sequencing, including far more accurate and quantitative records of splicing patterns. New studies with LRS datasets are being published at an exponential rate, generating a vast reservoir of information that can be leveraged to address a host of different research questions. However, mining such publicly available data in a tailored fashion is currently not easy, as the available software tools typically require familiarity with the command-line interface, which constitutes a significant obstacle to many researchers. Additionally, different research groups utilize different software packages to perform LRS analysis, which often prevents a direct comparison of published results across different studies. To address these challenges, we have developed the Long-Read Analysis Pipeline for Transcriptomics (L-RAPiT), a user-friendly, free pipeline requiring no dedicated computational resources or bioinformatics expertise. L-RAPiT can be implemented directly through Google Colaboratory, a system based on the open-source Jupyter notebook environment, and allows for the direct analysis of transcriptomic reads from Oxford Nanopore and PacBio LRS machines. This new pipeline enables the rapid, convenient, and standardized analysis of publicly available or newly generated LRS datasets.

Possibility and Challenges of Conversion of Current Virus Species Names to Linnaean Binomials.

Botanical, mycological, zoological, and prokaryotic species names follow the Linnaean format, consisting of an italicized Latinized binomen with a capitalized genus name and a lower case species epithet (e.g., Homo sapiens). Virus species names, however, do not follow a uniform format, and, even when binomial, are not Linnaean in style. In this thought exercise, we attempted to convert all currently official names of species included in the virus family Arenaviridae and the virus order Mononegavirales to Linnaean binomials, and to identify and address associated challenges and concerns. Surprisingly, this endeavor was not as complicated or time-consuming as even the authors of this article expected when conceiving the experiment. [Arenaviridae; binomials; ICTV; International Committee on Taxonomy of Viruses; Mononegavirales; virus nomenclature; virus taxonomy.].

The tale of the long tail: the cytoplasmic domain of HIV-1 gp41.

Envelope glycoproteins (Env) of lentiviruses typically possess unusually long cytoplasmic domains, often 150 amino acids or longer. It is becoming increasingly clear that these sequences contribute a diverse array of functional activities to the life cycle of their viruses. The cytoplasmic domain of gp41 (gp41CD) is required for replication of human immunodeficiency virus type 1 (HIV-1) in most but not all cell types, whereas it is largely dispensable for replication of simian immunodeficiency virus (SIV). Functionally, gp41CD has been shown to regulate rapid clathrin-mediated endocytosis of Env. The resultant low levels of Env expression at the cell surface likely serve as an immune avoidance mechanism to limit accessibility to the humoral immune response. Intracellular trafficking of Env is also regulated by gp41CD through interactions with a variety of cellular proteins. Furthermore, gp41CD has been implicated in the incorporation of Env into virions through an interaction with the virally encoded matrix protein. Most recently, the gp41CDs of HIV-1 and SIV were shown to activate the key cellular-transcription factor NF-κB via the serine/threonine kinase TAK1. Less well understood are the cytotoxicity- and apoptosis-inducing activities of gp41CD as well as potential roles in modulating the actin cytoskeleton and overcoming host cell restrictions. In this review, we summarize what is currently known about the cytoplasmic domains of HIV-1 and SIV and attempt to integrate the wealth of information in terms of defined functional activities.

Heterogenous expansion of polymorphonuclear myeloid-derived suppressor cells distinguishes high-risk sepsis immunophenotypes in Uganda.

BACKGROUND: Understanding of immune cell phenotypes associated with inflammatory and immunosuppressive host responses in sepsis is imprecise, particularly in low- and middle-income countries (LMICs), where the global sepsis burden is concentrated. In these settings, elucidation of immunophenotypes with prognostic importance is necessary to determine the relevance of emerging therapeutics and refine mechanistic investigations of sepsis immunopathology. METHODS: In a prospective cohort of adults hospitalized with suspected sepsis in Uganda (N = 43; median age 46 years [IQR 36-59], 24 [55.8%] living with HIV, 16 [37.2%] deceased at 60 days), we combined high-dimensional flow cytometry with unsupervised machine learning and manual gating to define peripheral immunophenotypes associated with increased risk of 60-day mortality. RESULTS: Patients who died showed heterogenous expansion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), with increased and decreased abundance of CD16negPD-L1dim and CD16brightPD-L1bright subsets, respectively, significantly associated with mortality. While differences between CD16negPD-L1dim cell abundance and mortality risk appeared consistent throughout the course of illness, those for the CD16brightPD-L1bright subset were more pronounced early after illness onset. Independent of HIV co-infection, depletion of CD4+ T cells, dendritic cells, and CD56-CD16bright NK cells were significantly associated with mortality risk, as was expansion of immature, CD56+CD16-CD11c+ NK cells. Abundance of T cells expressing inhibitory checkpoint proteins (PD-1, CTLA-4, LAG3) was similar between patients who died versus those who survived. CONCLUSIONS: This is the first study to define high risk immunophenotypes among adults with sepsis in sub-Saharan Africa, an immunologically distinct region where biologically informed treatment strategies are needed. More broadly, our findings highlight the clinical importance and complexity of MDSC expansion during sepsis and support emerging data that suggest a host-protective role for PD-L1 myeloid checkpoints in acute critical illness.

NF-κB subunits RelA and c-Rel selectively control CD4+ T cell function in multiple sclerosis and cancer.

The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.

COVID-19 immune signatures in Uganda persist in HIV co-infection and diverge by pandemic phase.

Little is known about the pathobiology of SARS-CoV-2 infection in sub-Saharan Africa, where severe COVID-19 fatality rates are among the highest in the world and the immunological landscape is unique. In a prospective cohort study of 306 adults encompassing the entire clinical spectrum of SARS-CoV-2 infection in Uganda, we profile the peripheral blood proteome and transcriptome to characterize the immunopathology of COVID-19 across multiple phases of the pandemic. Beyond the prognostic importance of myeloid cell-driven immune activation and lymphopenia, we show that multifaceted impairment of host protein synthesis and redox imbalance define core biological signatures of severe COVID-19, with central roles for IL-7, IL-15, and lymphotoxin-α in COVID-19 respiratory failure. While prognostic signatures are generally consistent in SARS-CoV-2/HIV-coinfection, type I interferon responses uniquely scale with COVID-19 severity in persons living with HIV. Throughout the pandemic, COVID-19 severity peaked during phases dominated by A.23/A.23.1 and Delta B.1.617.2/AY variants. Independent of clinical severity, Delta phase COVID-19 is distinguished by exaggerated pro-inflammatory myeloid cell and inflammasome activation, NK and CD8+ T cell depletion, and impaired host protein synthesis. Combining these analyses with a contemporary Ugandan cohort of adults hospitalized with influenza and other severe acute respiratory infections, we show that activation of epidermal and platelet-derived growth factor pathways are distinct features of COVID-19, deepening translational understanding of mechanisms potentially underlying SARS-CoV-2-associated pulmonary fibrosis. Collectively, our findings provide biological rationale for use of broad and targeted immunotherapies for severe COVID-19 in sub-Saharan Africa, illustrate the relevance of local viral and host factors to SARS-CoV-2 immunopathology, and highlight underemphasized yet therapeutically exploitable immune pathways driving COVID-19 severity.

Evidence against extracellular exposure of a highly immunogenic region in the C-terminal domain of the simian immunodeficiency virus gp41 transmembrane protein.

The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called "Kennedy epitope," that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia transmembrane protein with a single membrane-spanning domain and without any extracellular loops.

Transcriptomic analysis reveals that RasGEF1b deletion alters basal and LPS-induced expression of genes involved in chemotaxis and cytokine responses in macrophages.

Ras guanine nucleotide exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knock-out primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several genes with significantly different transcript levels between wild-type and knock-out macrophages. In total, 49 and 37 differentially expressed genes were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to down-regulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that the expression of genes identified as down-regulated after LPS stimulation was also decreased in the knock-out cells under basal conditions. We used a luciferase-based reporter assay to showcase the capability of RasGEF1b in activating the Serpinb2 promoter. Notably, knockdown of RasGEF1b in RAW264.7 macrophages resulted in impaired transcriptional activation of the Serpinb2 promoter, both in constitutive and LPS-stimulated conditions. This study provides a small collection of genes that shows relative expression changes effected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and propose that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages.

Brief Report: Detection of Urine Lipoarabinomannan Is Associated With Proinflammatory Innate Immune Activation, Impaired Host Defense, and Organ Dysfunction in Adults With Severe HIV-Associated Tuberculosis in Uganda.

BACKGROUND: The immunopathology of disseminated HIV-associated tuberculosis (HIV/TB), a leading cause of critical illness and death among persons living with HIV in sub-Saharan Africa, is incompletely understood. Reflective of hematogenously disseminated TB, detection of lipoarabinomannan (LAM) in urine is associated with greater bacillary burden and poor outcomes in adults with HIV/TB. METHODS: We determined the relationship between detection of urine TB-LAM, organ dysfunction, and host immune responses in a prospective cohort of adults hospitalized with severe HIV/TB in Uganda. Generalized additive models were used to analyze the association between urine TB-LAM grade and concentrations of 14 soluble immune mediators. Whole-blood RNA-sequencing data were used to compare transcriptional profiles between patients with high- vs. low-grade TB-LAM results. RESULTS: Among 157 hospitalized persons living with HIV, 40 (25.5%) had positive urine TB-LAM testing. Higher TB-LAM grade was associated with more severe physiologic derangement, organ dysfunction, and shock. Adjusted generalized additive models showed that higher TB-LAM grade was significantly associated with higher concentrations of mediators reflecting proinflammatory innate and T-cell activation and chemotaxis (IL-8, MIF, MIP-1ß/CCL4, and sIL-2Ra/sCD25). Transcriptionally, patients with higher TB-LAM grades demonstrated multifaceted impairment of antibacterial defense including reduced expression of genes encoding cytotoxic and autophagy-related proteins and impaired cross-talk between innate and cell-mediated immune effectors. CONCLUSIONS: Our findings add to emerging data suggesting pathobiological relationships between LAM, TB dissemination, innate cell activation, and evasion of host immunity in severe HIV/TB. Further translational studies are needed to elucidate the role for immunomodulatory therapies, in addition to optimized anti-TB treatment, in this often critically ill population.

HIV infection drives pro-inflammatory immunothrombotic pathway activation and organ dysfunction among adults with sepsis in Uganda.

BACKGROUND: The global burden of sepsis is concentrated in high HIV-burden settings in sub-Saharan Africa (SSA). Despite this, little is known about the immunopathology of sepsis in persons with HIV (PWH) in the region. We sought to determine the influence of HIV on host immune responses and organ dysfunction among adults hospitalized with suspected sepsis in Uganda. DESIGN: Prospective cohort study. METHODS: We compared organ dysfunction and 30-day outcome profiles of PWH and those without HIV. We quantified 14 soluble immune mediators, reflective of key domains of sepsis immunopathology, and performed whole-blood RNA-sequencing on samples from a subset of patients. We used propensity score methods to match PWH and those without HIV by demographics, illness duration, and clinical severity, and compared immune mediator concentrations and gene expression profiles across propensity score-matched groups. RESULTS: Among 299 patients, 157 (52.5%) were PWH (clinical stage 3 or 4 in 80.3%, 67.7% with known HIV on antiretroviral therapy). PWH presented with more severe physiologic derangement and shock, and had higher 30-day mortality (34.5% vs. 10.2%; P  < 0.001). Across propensity score-matched groups, PWH exhibited greater pro-inflammatory immune activation, including upregulation of interleukin (IL)-6, IL-8, IL-15, IL-17 and HMGB1 signaling, with concomitant T-cell exhaustion, prothrombotic pathway activation, and angiopoeitin-2-related endothelial dysfunction. CONCLUSIONS: Sepsis-related organ dysfunction and mortality in Uganda disproportionately affect PWH, who demonstrate exaggerated activation of multiple immunothrombotic and metabolic pathways implicated in sepsis pathogenesis. Further investigations are needed to refine understanding of sepsis immunopathology in PWH, particularly mechanisms amenable to therapeutic manipulation.

A most versatile kinase: The catalytic subunit of PKA in T-cell biology.

The cAMP-dependent protein kinase, more commonly referred to as protein kinase A (PKA), is one of the most-studied enzymes in biology. PKA is ubiquitously expressed in mammalian cells, can be activated in response to a plethora of biological stimuli, and phosphorylates more than 250 known substrates. Indeed, PKA is of central importance to a wide range of organismal processes, including energy homeostasis, memory formation and immunity. It serves as the primary effector of the second-messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP), which is believed to have mostly inhibitory effects on the adaptive immune response. In particular, elevated levels of intracellular cAMP inhibit the activation of conventional T cells by limiting signal transduction through the T-cell receptor and altering gene expression, primarily in a PKA-dependent manner. Regulatory T cells have been shown to increase the cAMP levels in adjacent T cells by direct and indirect means, but the role of cAMP within regulatory T cells themselves remains incompletely understood. Paradoxically, cAMP has been implicated in promoting T-cell activation as well, adding another functional dimension beyond its established immunosuppressive effects. Furthermore, PKA can phosphorylate the NF-κB subunit p65, a transcription factor that is essential for T-cell activation, independently of cAMP. This phosphorylation of p65 drastically enhances NF-κB-dependent transcription and thus is likely to facilitate immune activation. How these immunosuppressive and immune-activating properties of PKA balance in vivo remains to be elucidated. This review provides a brief overview of PKA regulation, its ability to affect NF-κB activation, and its diverse functions in T-cell biology.

Charting protein dephosphorylation triggered by Toll-like receptor (TLR) signaling in macrophages and its role in health and disease.

Toll-like receptor (TLR) signaling induces substantial changes in the phosphoproteome of innate immune cells, mainly in the form of increased phosphorylation of signaling intermediaries. Loss of constitutive phosphorylation occurs simultaneously, but these transitions from a stable, phosphorylated state in resting cells to a sustained underphosphorylated state in activated cells have received far less attention. This review provides an overview of phosphorylation sites downregulated during TLR-mediated signaling, with a particular focus on TLR4 activation by lipopolysaccharide (LPS). Energy homeostasis, the cell cycle, mitochondrial fission, and gene regulation are among the biological events in macrophages that are regulated through the downregulation of phosphoproteins as part of intracellular signaling events. Phosphoproteomics studies on innate immune cells have identified hundreds of hitherto uncharacterized phosphorylation sites that are lost upon stimulation, indicating that protein hypophosphorylation is a significant, largely unexplored layer of complexity in the TLR4 pathway.

Metformin selectively dampens the acute inflammatory response through an AMPK-dependent mechanism.

Metformin is a first-line drug in the treatment of type-2 diabetes mellitus (T2DM). In addition to its antigluconeogenic and insulin-sensitizing properties, metformin has emerged as a potent inhibitor of the chronic inflammatory response of macrophages. In particular, metformin treatment has been shown to reduce expression of interleukin (IL-) 1ß during long-term exposure to the pro-inflammatory stimulus lipopolysaccharide (LPS) through a reduction in reactive oxygen species (ROS), which decreases the levels of the hypoxia-inducible factor (HIF) 1-α, and through enhanced expression of IL-10. However, the effect of metformin on the acute inflammatory response, before significant levels of ROS accumulate in the cell, has not been explored. Here, we show that metformin alters the acute inflammatory response through its activation of AMP-activated protein kinase (AMPK), but independently of HIF1-α and IL-10, in primary macrophages and two macrophage-like cell lines. Thus, metformin changes the acute and the chronic inflammatory response through fundamentally distinct mechanisms. Furthermore, RNA-seq analysis reveals that metformin pretreatment affects the levels of a large yet selective subset of inflammatory genes, dampening the response to short-term LPS exposure and affecting a wide range of pathways and biological functions. Taken together, these findings reveal an unexpected complexity in the anti-inflammatory properties of this widely used drug.