Immunity to the microbiota promotes sensory neuron regeneration.

Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.

Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair.

Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.

Second signals rescue B cells from activation-induced mitochondrial dysfunction and death.

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response-activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

Extracellular Acidity Reprograms Macrophage Metabolism and Innate Responsiveness.

Although organ hypofunction and immunosuppression are life-threatening features of severe sepsis, the hypofunctioning organs and immune cells usually regain normal functionality if patients survive. Because tissue interstitial fluid can become acidic during the septic response, we tested the hypothesis that low extracellular pH (pHe) can induce reversible metabolic and functional changes in peritoneal macrophages from C57BL/6J mice. When compared with macrophages cultured at normal pHe, macrophages living in an acidic medium used less glucose and exogenous fatty acid to produce ATP. Lactate, glutamine, and de novo-synthesized fatty acids supported ATP production by mitochondria that gained greater mass, maximal oxygen consumption rate, and spare respiratory capacity. The cells transitioned to an M2-like state, with altered immune responses to LPS and slightly decreased phagocytic ability, yet they regained basal energy production, normal mitochondrial function, and proinflammatory responsiveness when neutral pHe was restored. Low pHe induces changes that support macrophage survival while rendering the cells less proinflammatory (more "tolerant") and less able to phagocytose bacteria. Macrophage responses to low interstitial pH may contribute to the reversible organ hypofunction and immunoparalysis noted in many patients with sepsis.

Immunity to commensal skin fungi promotes psoriasiform skin inflammation.

Under steady-state conditions, the immune system is poised to sense and respond to the microbiota. As such, immunity to the microbiota, including T cell responses, is expected to precede any inflammatory trigger. How this pool of preformed microbiota-specific T cells contributes to tissue pathologies remains unclear. Here, using an experimental model of psoriasis, we show that recall responses to commensal skin fungi can significantly aggravate tissue inflammation. Enhanced pathology caused by fungi preexposure depends on Th17 responses and neutrophil extracellular traps and recapitulates features of the transcriptional landscape of human lesional psoriatic skin. Together, our results propose that recall responses directed to skin fungi can directly promote skin inflammation and that exploration of tissue inflammation should be assessed in the context of recall responses to the microbiota.

Eosinophils Do Not Drive Acute Muscle Pathology in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Eosinophils are present in muscle lesions associated with Duchenne muscular dystrophy and dystrophin-deficient mdx mice that phenocopy this disorder. Although it has been hypothesized that eosinophils promote characteristic inflammatory muscle damage, this has not been fully examined. In this study, we generated mice with the dystrophin mutation introduced into PHIL, a strain with a transgene that directs lineage-specific eosinophil ablation. We also explored the impact of eosinophil overabundance on dystrophinopathy by introducing the dystrophin mutation into IL-5 transgenic mice. We evaluated the degree of eosinophil infiltration in association with myofiber size distribution, centralized nuclei, serum creatine kinase, and quantitative histopathology scores. Among our findings, eosinophils were prominent in the quadriceps muscles of 4-wk-old male mdx mice but no profound differences were observed in the quantitative measures of muscle damage when comparing mdx versus mdx.PHIL versus mdx.IL5tg mice, despite dramatic differences in eosinophil infiltration (CD45+CD11c-Gr1-MHC class IIloSiglecF+ eosinophils at 1.2 ± 0.34% versus <0.1% versus 20 ± 7.6% of total cells, respectively). Further evaluation revealed elevated levels of eosinophil chemoatttractants eotaxin-1 and RANTES in the muscle tissue of all three dystrophin-deficient strains; eotaxin-1 concentration in muscle correlated inversely with age. Cytokines IL-4 and IL-1R antagonist were also detected in association with eosinophils in muscle. Taken together, our findings challenge the long-held perception of eosinophils as cytotoxic in dystrophin-deficient muscle; we show clearly that eosinophil infiltration is not a driving force behind acute muscle damage in the mdx mouse strain. Ongoing studies will focus on the functional properties of eosinophils in this unique microenvironment.

Increased Mitochondrial Biogenesis and Reactive Oxygen Species Production Accompany Prolonged CD4+ T Cell Activation.

Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

Influenza NS1 directly modulates Hedgehog signaling during infection.

The multifunctional NS1 protein of influenza A viruses suppresses host cellular defense mechanisms and subverts other cellular functions. We report here on a new role for NS1 in modifying cell-cell signaling via the Hedgehog (Hh) pathway. Genetic epistasis experiments and FRET-FLIM assays in Drosophila suggest that NS1 interacts directly with the transcriptional mediator, Ci/Gli1. We further confirmed that Hh target genes are activated cell-autonomously in transfected human lung epithelial cells expressing NS1, and in infected mouse lungs. We identified a point mutation in NS1, A122V, that modulates this activity in a context-dependent fashion. When the A122V mutation was incorporated into a mouse-adapted influenza A virus, it cell-autonomously enhanced expression of some Hh targets in the mouse lung, including IL6, and hastened lethality. These results indicate that, in addition to its multiple intracellular functions, NS1 also modifies a highly conserved signaling pathway, at least in part via cell autonomous activities. We discuss how this new Hh modulating function of NS1 may influence host lethality, possibly through controlling cytokine production, and how these new insights provide potential strategies for combating infection.

Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands.

CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. In this study, we examined the ability of MLN7243, a recently described ubiquitin-activating enzyme E1 inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus-encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines and can profoundly reduce the generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I-restricted Ag processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, ubiquitin-dependent and -independent pathways robustly contribute to MHC class I-based immunosurveillance.

Novel signal transducer and activator of transcription 1 mutation disrupts small ubiquitin-related modifier conjugation causing gain of function.

BACKGROUND: Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein function. OBJECTIVE: We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). METHODS: STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. RESULTS: We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants. CONCLUSION: This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects.

Emergence of Polyfunctional Cytotoxic CD4+ T Cells in Mycobacterium avium Immune Reconstitution Inflammatory Syndrome in Human Immunodeficiency Virus-Infected Patients.

Background: Immune reconstitution inflammatory syndrome (IRIS) is an aberrant inflammatory response in individuals with advanced human immunodeficiency virus (HIV) infection, after antiretroviral therapy (ART) initiation. The pathogenesis of Mycobacterium avium complex (MAC)-associated IRIS has not been fully elucidated. Methods: We investigated monocyte and CD4+ T-cell responses in vitro, tumor necrosis factor (TNF) expression in tissues, and plasma cytokines and inflammatory markers, in 13 HIV-infected patients with MAC-IRIS and 14 HIV-uninfected patients with pulmonary MAC infection. Results: Prior to ART, HIV-infected compared with HIV-uninfected patients, had reduced TNF+ monocytes (P = .013), although similar cytokine (interferon gamma [IFN-γ], TNF, interleukin 2 [IL-2], and interleukin 17 [IL-17])-expressing CD4+ T cells. During IRIS, monocyte cytokine production was restored. IFN-γ+ (P = .027), TNF+ (P = .004), and polyfunctional CD4+ T cells (P = 0.03) also increased. These effectors were T-betlow, and some expressed markers of degranulation and cytotoxic potential. Blockade of cytotoxic T-lymphocyte associated protein 4 and lymphocyte activation gene-3 further increased CD4+ T-cell cytokine production. Tissue immunofluorescence showed higher proportions of CD4+ and CD68+ (monocyte/macrophage) cells expressed TNF during IRIS compared with HIV-uninfected patients. Plasma IFN-γ (P = .048), C-reactive protein (P = .008), and myeloperoxidase (P < .001) levels also increased, whereas interleukin 10 decreased (P = .008) during IRIS. Conclusions: Advanced HIV infection was associated with impaired MAC responses. Restoration of monocyte responses and expansion of polyfunctional MAC-specific T-betlow CD4+ T cells with cytotoxic potential after ART initiation may overwhelm existing regulatory and inhibitory mechanisms, leading to MAC-IRIS.

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread.

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

Early Intracellular Trafficking of Granulibacter bethesdensis in Human Macrophages.

Granulibacter bethesdensis is a Gram-negative bacterium that infects patients with chronic granulomatous disease (CGD), a primary immunodeficiency marked by a defect in NOX2, the phagocyte NADPH oxidase. Previous studies have shown that NOX2 is essential for killing of G. bethesdensis by neutrophils and monocytes and that the bacteriostatic activity of monocyte-derived macrophages (MDM) requires NOX2 and gamma interferon (IFN-γ) pretreatment. To determine whether G. bethesdensis evades phagolysosomal killing, a host defense pathway intact in both normal and CGD MDM, or whether it occupies a distinct intracellular niche in CGD MDM, we assessed the trafficking patterns of this organism. We observed colocalization of G. bethesdensis with an early endosome antigen 1 (EEA1)-positive compartment, followed by colocalization with lysosome-associated membrane protein 1 (LAMP1)-positive and LysoTracker-positive late phagosomes; these characteristics were similar in both normal and CGD MDM. Despite localization to acidified late phagosomes, viable G. bethesdensis cells were recovered from viable MDM in numbers greater than in the initial input up to 6 days after infection. G. bethesdensis remains, and in some cases appears to divide, within a membrane-bound compartment for the entire 6-day time course. These findings indicate that this organism resists both oxygen-dependent and oxygen-independent phagolysosomal antimicrobial systems of human macrophages.

The small GTPase RAB-11 directs polarized exocytosis of the intracellular pathogen N. parisii for fecal-oral transmission from C. elegans.

Pathogen exit is a key stage in the spread and propagation of infectious disease, with the fecal-oral route being a common mode of disease transmission. However, it is poorly understood which molecular pathways provide the major modes for intracellular pathogen exit and fecal-oral transmission in vivo. Here, we use the transparent nematode Caenorhabditis elegans to investigate intestinal cell exit and fecal-oral transmission by the natural intracellular pathogen Nematocida parisii, which is a recently identified species of microsporidia. We show that N. parisii exits from polarized host intestinal cells by co-opting the host vesicle trafficking system and escaping into the lumen. Using a genetic screen, we identified components of the host endocytic recycling pathway that are required for N. parisii spore exit via exocytosis. In particular, we show that the small GTPase RAB-11 localizes to apical spores, is required for spore-containing compartments to fuse with the apical plasma membrane, and is required for spore exit. In addition, we find that RAB-11-deficient animals exhibit impaired contagiousness, supporting an in vivo role for this host trafficking factor in microsporidia disease transmission. Altogether, these findings provide an in vivo example of the major mode of exit used by a natural pathogen for disease spread via fecal-oral transmission.

Ubiquitin-mediated response to microsporidia and virus infection in C. elegans.

Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host C. elegans to show an in vivo role for ubiquitin-mediated response to the microsporidian species Nematocida parisii, as well to the Orsay virus, another natural intracellular pathogen of C. elegans. We analyze gene expression of C. elegans in response to N. parisii, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog cul-6, which we show is important for ubiquitin targeting of N. parisii cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against N. parisii infection. We also find that SCF ligase components like cul-6 promote defense against viral infection, where they have a more robust role than against N. parisii infection. This difference may be due to suppression of the host ubiquitylation system by N. parisii: when N. parisii is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by N. parisii and virus may be perturbation of the UPS. Altogether, our results demonstrate an in vivo role for ubiquitin-mediated defense against microsporidian and viral infections in C. elegans.

Phosphoproteome profiling of the macrophage response to different toll-like receptor ligands identifies differences in global phosphorylation dynamics.

Toll-like receptors (TLRs) are among the first sensors that detect infection and drive immune response. Macrophages encountering a pathogen are usually stimulated not by one TLR, but by a combination of TLRs engaged by distinct microbe ligands. To understand the integrated signaling under complex conditions, we investigated the differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using a single responding cell population. We performed a global, quantitative, early poststimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance the understanding of how macrophages sense and respond to a diverse set of TLR stimuli.

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo . Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statement: When cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo . Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

Myristoylated, Alanine-rich C-kinase Substrate (MARCKS) regulates Toll-like receptor 4 signaling in macrophages.

MARCKS (Myristoylated Alanine-rich C-kinase Substrate) is a membrane protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (Toll-like receptor 4). The presence of MARCKS and the formation of phospho-MARCKS in macrophages have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. To investigate the role of MARCKS during inflammation, we activated macrophages using LPS with or without the addition of a PKC inhibitor. We found that PKC inhibition substantially decreased macrophage IL6 and TNF cytokine production. In addition, confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation. CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated TNF and IL6 production, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes and signaling pathways, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory related diseases.