Erratum: Update Notice: Computational Analysis of Plasma Lipidomics from Mice Fed Standard Chow and Ketogenic Diet.

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Computational Analysis of Plasma Lipidomics from Mice Fed Standard Chow and Ketogenic Diet.

Dietary saturated fatty acids (SFAs) are upregulated in the blood circulation following digestion. A variety of circulating lipid species have been implicated in metabolic and inflammatory diseases; however, due to the extreme variability in serum or plasma lipid concentrations found in human studies, established reference ranges are still lacking, in addition to lipid specificity and diagnostic biomarkers. Mass spectrometry is widely used for identification of lipid species in the plasma, and there are many differences in sample extraction methods within the literature. We used ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS) to compare relative peak abundance of specific lipid species within the following lipid classes: free fatty acids (FFAs), triglycerides (TAGs), phosphatidylcholines (PCs), and sphingolipids (SGs), in the plasma of mice fed a standard chow (SC; low in SFAs) or ketogenic diet (KD; high in SFAs) for two weeks. In this protocol, we used Principal Component Analysis (PCA) and R to visualize how individual mice clustered together according to their diet, and we found that KD-fed mice displayed unique blood profiles for many lipid species identified within each lipid class compared to SC-fed mice. We conclude that two weeks of KD feeding is sufficient to significantly alter circulating lipids, with PCs being the most altered lipid class, followed by SGs, TAGs, and FFAs, including palmitic acid (PA) and PA-saturated lipids. This protocol is needed to advance knowledge on the impact that SFA-enriched diets have on concentrations of specific lipids in the blood that are known to be associated with metabolic and inflammatory diseases. Key features • Analysis of relative plasma lipid concentrations from mice on different diets using R. • Lipidomics data collected via ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS). • Allows for a comprehensive comparison of diet-dependent plasma lipid profiles, including a variety of specific lipid species within several different lipid classes. • Accumulation of certain free fatty acids, phosphatidylcholines, triglycerides, and sphingolipids are associated with metabolic and inflammatory diseases, and plasma concentrations may be clinically useful.

A new frontier for fat: dietary palmitic acid induces innate immune memory.

Dietary saturated fats have recently been appreciated for their ability to modify innate immune cell function, including monocytes, macrophages, and neutrophils. Many dietary saturated fatty acids (SFAs) embark on a unique pathway through the lymphatics following digestion, and this makes them intriguing candidates for inflammatory regulation during homeostasis and disease. Specifically, palmitic acid (PA) and diets enriched in PA have recently been implicated in driving innate immune memory in mice. PA has been shown to induce long-lasting hyper-inflammatory capacity against secondary microbial stimuli in vitro and in vivo, and PA-enriched diets alter the developmental trajectory of stem cell progenitors in the bone marrow. Perhaps the most relevant finding is the ability of exogenous PA to enhance clearance of fungal and bacterial burdens in mice; however, the same PA treatment enhances endotoxemia severity and mortality. Westernized countries are becoming increasingly dependent on SFA-enriched diets, and a deeper understanding of SFA regulation of innate immune memory is imperative in this pandemic era.

Enriched dietary saturated fatty acids induce trained immunity via ceramide production that enhances severity of endotoxemia and clearance of infection.

Trained immunity is an innate immune memory response that is induced by a primary inflammatory stimulus that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Dietary fatty acids can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli to induce trained immunity. Here we find mice fed a diet enriched exclusively in saturated fatty acids (ketogenic diet; KD) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and hyperglycemia. We find KD alters the composition of the hematopoietic stem cell compartment and enhances the response of bone marrow macrophages, monocytes, and splenocytes to secondary LPS challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in KD-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA induces a hyper-inflammatory response to LPS in macrophages, and this was dependent on the synthesis of ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality in response to systemic LPS, and this phenotype was not reversible for up to 7 days post-PA-exposure. Conversely, we find PA exposure enhanced clearance of Candida albicans in Rag1-/- mice. Lastly, we show that oleic acid, which depletes intracellular ceramide, reverses PA-induced hyper-inflammation in macrophages and enhanced mortality in response to LPS. These implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory and highlight the plasticity of this innate immune reprogramming by dietary constituents.

C3aR plays both sides in regulating resistance to bacterial infections.

Activation of the complement pathway results in the production of bioactive C3a, a product of C3 cleavage, which interacts with membrane-bound receptor C3aR to regulate innate immune cell function and outcome of bacterial infection. Specifically, previous research has identified mechanistically distinct and cell type-specific roles for C3aR in regulating innate immune cell inflammatory state, antimicrobial killing capacity, and metabolism. Historically, the production of C3a has been relegated to the serum; however, recent studies have provided evidence that various cell types can produce intracellular C3a that stimulates intracellular C3aR. In light of these new results, it is imperative that we revisit previous studies regarding the role of C3aR in controlling bacterial infections and analyze these results in the context of both extracellular and intracellular C3a production and C3aR activation. Thus, this review will cover specific roles of C3aR in driving cell type-specific and tissue specific responses during bacterial infections and emphasize the contribution of the C3a-C3aR axis in regulating host resistance to bacterial infection.

TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease-associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. Here, we report that TREM2 is a thyroid hormone-regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone and synthetic thyroid hormone agonists (thyromimetics). Our findings report the endocrine regulation of TREM2 by thyroid hormone, and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small-molecule therapeutic agents.

TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. We report here that TREM2 is a thyroid hormone regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone. Both endogenous thyroid hormone and sobetirome, a synthetic thyroid hormone agonist drug, suppress pro-inflammatory cytokine production from myeloid cells including macrophages that have been treated with the SARS-CoV-2 spike protein which produces a strong, pro-inflammatory phenotype. Thyroid hormone agonism was also found to induce phagocytic behavior in microglia, a phenotype consistent with activation of the TREM2 pathway. The thyroid hormone antagonist NH-3 blocks the anti-inflammatory effects of thyroid hormone agonists and suppresses microglia phagocytosis. Finally, in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model, treatment with Sob-AM2, a CNS-penetrating sobetirome prodrug, results in increased Trem2 expression in disease lesion resident myeloid cells which correlates with therapeutic benefit in the EAE clinical score and reduced damage to myelin. Our findings represent the first report of endocrine regulation of TREM2 and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small molecule therapeutic agents.

A Rapid Caspase-11 Response Induced by IFNγ Priming Is Independent of Guanylate Binding Proteins.

In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFNγ priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFNγ treatment. Indeed, IFNγ priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFNγ priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFNγ.

Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy.

Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart. METHODS: H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction. RESULTS: LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality. CONCLUSIONS: Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.

Western diet regulates immune status and the response to LPS-driven sepsis independent of diet-associated microbiome.

Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library.

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

Neurotoxic reactive astrocytes are induced by activated microglia.

Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1α, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.

Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity.

Cell death and release of proinflammatory mediators contribute to mortality during sepsis. Specifically, caspase-11-dependent cell death contributes to pathology and decreases in survival time in sepsis models. Priming of the host cell, through TLR4 and interferon receptors, induces caspase-11 expression, and cytosolic LPS directly stimulates caspase-11 activation, promoting the release of proinflammatory cytokines through pyroptosis and caspase-1 activation. Using a CRISPR-Cas9-mediated genome-wide screen, we identified novel mediators of caspase-11-dependent cell death. We found a complement-related peptidase, carboxypeptidase B1 (Cpb1), to be required for caspase-11 gene expression and subsequent caspase-11-dependent cell death. Cpb1 modifies a cleavage product of C3, which binds to and activates C3aR, and then modulates innate immune signaling. We find the Cpb1-C3-C3aR pathway induces caspase-11 expression through amplification of MAPK activity downstream of TLR4 and Ifnar activation, and mediates severity of LPS-induced sepsis (endotoxemia) and disease outcome in mice. We show C3aR is required for up-regulation of caspase-11 orthologues, caspase-4 and -5, in primary human macrophages during inflammation and that c3aR1 and caspase-5 transcripts are highly expressed in patients with severe sepsis; thus, suggesting that these pathways are important in human sepsis. Our results highlight a novel role for complement and the Cpb1-C3-C3aR pathway in proinflammatory signaling, caspase-11 cell death, and sepsis severity.

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

A PmrB-Regulated Deacetylase Required for Lipid A Modification and Polymyxin Resistance in Acinetobacter baumannii.

Emerging resistance to "last-resort" polymyxin antibiotics in Gram-negative bacteria is a significant threat to public health. We identified the Acinetobacter baumannii NaxD deacetylase as a critical mediator of lipid A modification resulting in polymyxin resistance and demonstrated that naxD is regulated by the sensor kinase PmrB. This represents the first description of a specific PmrB-regulated gene contributing to polymyxin resistance in A. baumannii and highlights NaxD as a putative drug target to reverse polymyxin resistance.

Low doses of imatinib induce myelopoiesis and enhance host anti-microbial immunity.

Imatinib mesylate (Gleevec) inhibits Abl1, c-Kit, and related protein tyrosine kinases (PTKs) and serves as a therapeutic for chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib also has efficacy against various pathogens, including pathogenic mycobacteria, where it decreases bacterial load in mice, albeit at doses below those used for treating cancer. We report that imatinib at such low doses unexpectedly induces differentiation of hematopoietic stem cells and progenitors in the bone marrow, augments myelopoiesis but not lymphopoiesis, and increases numbers of myeloid cells in blood and spleen. Whereas progenitor differentiation relies on partial inhibition of c-Kit by imatinib, lineage commitment depends upon inhibition of other PTKs. Thus, imatinib mimics "emergency hematopoiesis," a physiological innate immune response to infection. Increasing neutrophil numbers by adoptive transfer sufficed to reduce mycobacterial load, and imatinib reduced bacterial load of Franciscella spp., which do not utilize imatinib-sensitive PTKs for pathogenesis. Thus, potentiation of the immune response by imatinib at low doses may facilitate clearance of diverse microbial pathogens.

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.