Establishing thresholds for cytokine storm and defining their relationship to disease severity in respiratory viral infections.

Previous studies have identified cytokines associated with respiratory virus infection illness outcome. However, few studies have included comprehensive cytokine panels, longitudinal analyses, and/or simultaneous assessment across the severity spectrum. This, coupled with subjective definitions of cytokine storm syndrome (CSS), have contributed to inconsistent findings of cytokine signatures, particularly with COVID severity. Here, we measured 38 plasma cytokines and compared profiles in healthy, SARS-CoV-2 infected, and multisystem inflammatory syndrome in children (MIS-C) patients (n = 169). Infected patients spanned the severity spectrum and were classified as Asymptomatic, Mild, Moderate or Severe. Our results showed acute cytokine profiles and longitudinal dynamics of IL1Ra, IL10, MIP1b, and IP10 can differentiate COVID severity groups. Only 4% of acutely infected patients exhibited hypercytokinemia. Of these subjects, 3 were Mild, 3 Moderate, and 1 Severe, highlighting the lack of association between CSS and COVID severity. Additionally, we identified IL1Ra and TNFa as potential biomarkers for patients at high risk for long COVID. Lastly, we compare hypercytokinemia profiles across COVID and influenza patients and show distinct elevated cytokine signatures, wherein influenza induces the most elevated cytokine profile. Together, these results identify key analytes that, if obtained at early time points, can predict COVID illness outcome and/or risk of complications, and provide novel insight for improving the conceptual framework of hypercytokinemia, wherein CSS is a subgroup that requires concomitant severe clinical manifestations, and including a list of cytokines that can distinguish between subtypes of hypercytokinemia.

Group IIA secreted phospholipase A2 (PLA2G2A) augments adipose tissue thermogenesis.

Group IIA secreted phospholipase A2 (PLA2G2A) hydrolyzes glycerophospholipids at the sn-2 position resulting in the release of fatty acids and lysophospholipids. C57BL/6 mice do not express Pla2g2a due to a frameshift mutation (wild-type [WT] mice). We previously reported that transgenic expression of human PLA2G2A in C57BL/6 mice (IIA+ mice) protects against weight gain and insulin resistance, in part by increasing total energy expenditure. Additionally, we found that brown and white adipocytes from IIA+ mice have increased expression of mitochondrial uncoupling markers, such as uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor-gamma coactivator, and PR domain containing 16, suggesting that the energy expenditure phenotype might be due to an increased thermogenic capacity in adipose tissue. Here, we further characterize the impact of PLA2G2A on thermogenic mechanisms in adipose tissue. Metabolic analysis of WT and IIA+ mice revealed that even when housed within their thermoneutral zone, IIA+ mice have elevated energy expenditure compared to WT littermates. Increased energy expenditure in IIA+ mice is associated with increased citrate synthase activity in brown adipose tissue (BAT) and increased mitochondrial respiration in both brown and white adipocytes. We also observed that direct addition of recombinant PLA2G2A enzyme to in vitro cultured adipocytes results in the marked induction of UCP1 protein expression. Finally, we report that PLA2G2A induces the expression of numerous transcripts related to energy substrate transport and metabolism in BAT, suggestive of an increase in substrate flux to fuel BAT activity. These data demonstrate that PLA2G2A enhances adipose tissue thermogenesis, in part, through elevated substrate delivery and increased mitochondrial content in BAT.

Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells.

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.

Dynamic metabolic reprogramming in dendritic cells: An early response to influenza infection that is essential for effector function.

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.

Influenza A virus directly modulates mouse eosinophil responses.

Allergic asthma and influenza are common respiratory diseases with a high probability of co-occurrence. During the 2009 influenza pandemic, hospitalized patients with influenza experienced lower morbidity if asthma was an underlying condition. We have previously demonstrated that acute allergic asthma protects mice from severe influenza and have implicated eosinophils in the airways of mice with allergic asthma as participants in the antiviral response. However, very little is known about how eosinophils respond to direct exposure to influenza A virus (IAV) or the microenvironment in which the viral burden is high. We hypothesized that eosinophils would dynamically respond to the presence of IAV through phenotypic, transcriptomic, and physiologic changes. Using our mouse model of acute fungal asthma and influenza, we showed that eosinophils in lymphoid tissues were responsive to IAV infection in the lungs and altered surface expression of various markers necessary for cell activation in a niche-specific manner. Siglec-F expression was altered in a subset of eosinophils after virus exposure, and those expressing high Siglec-F were more active (IL-5Rαhi CD62Llo ). While eosinophils exposed to IAV decreased their overall transcriptional activity and mitochondrial oxygen consumption, transcription of genes encoding viral recognition proteins, Ddx58 (RIG-I), Tlr3, and Ifih1 (MDA5), were up-regulated. CD8+ T cells from IAV-infected mice expanded in response to IAV PB1 peptide-pulsed eosinophils, and CpG methylation in the Tbx21 promoter was reduced in these T cells. These data offer insight into how eosinophils respond to IAV and help elucidate alternative mechanisms by which they regulate antiviral immune responses during IAV infection.

Fueling influenza and the immune response: Implications for metabolic reprogramming during influenza infection and immunometabolism.

Recent studies support the notion that glycolysis and oxidative phosphorylation are rheostats in immune cells whose bioenergetics have functional outputs in terms of their biology. Specific intrinsic and extrinsic molecular factors function as molecular potentiometers to adjust and control glycolytic to respiratory power output. In many cases, these potentiometers are used by influenza viruses and immune cells to support pathogenesis and the host immune response, respectively. Influenza virus infects the respiratory tract, providing a specific environmental niche, while immune cells encounter variable nutrient concentrations as they migrate in response to infection. Immune cell subsets have distinct metabolic programs that adjust to meet energetic and biosynthetic requirements to support effector functions, differentiation, and longevity in their ever-changing microenvironments. This review details how influenza coopts the host cell for metabolic reprogramming and describes the overlap of these regulatory controls in immune cells whose function and fate are dictated by metabolism. These details are contextualized with emerging evidence of the consequences of influenza-induced changes in metabolic homeostasis on disease progression.

Myeloid Slc2a1-Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT1.

Macrophages (MΦs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MΦ activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MΦs (BMDM) from Slc2a1M-/- mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MΦ lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1M-/- BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro- and anti-inflammatory markers, yet less oxidative stress. Slc2a1M-/- BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MΦs of lean Slc2a1M-/- mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr-/- mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1M-/- BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MΦ were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MΦ function in chronic diseases.

Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention.

Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.

Pharmacologic activation of estrogen receptor ß increases mitochondrial function, energy expenditure, and brown adipose tissue.

Most satiety-inducing obesity therapeutics, despite modest efficacy, have safety concerns that underscore the need for effective peripherally acting drugs. An attractive therapeutic approach for obesity is to optimize/maximize energy expenditure by increasing energy-utilizing thermogenic brown adipose tissue. We used in vivo and in vitro models to determine the role of estrogen receptor ß (ER-ß) and its ligands on adipose biology. RNA sequencing and metabolomics were used to determine the mechanism of action of ER-ß and its ligands. Estrogen receptor ß (ER-ß) and its selective ligand reprogrammed preadipocytes and precursor stem cells into brown adipose tissue and increased mitochondrial respiration. An ER-ß-selective ligand increased markers of tricarboxylic acid-dependent and -independent energy biogenesis and oxygen consumption in mice without a concomitant increase in physical activity or food consumption, all culminating in significantly reduced weight gain and adiposity. The antiobesity effects of ER-ß ligand were not observed in ER-ß-knockout mice. Serum metabolite profiles of adult lean and juvenile mice were comparable, while that of adult obese mice was distinct, indicating a possible impact of obesity on age-dependent metabolism. This phenotype was partially reversed by ER-ß-selective ligand. These data highlight a new role for ER-ß in adipose biology and its potential to be a safer alternative peripheral therapeutic target for obesity.-Ponnusamy, S., Tran, Q. T., Harvey, I., Smallwood, H. S., Thiyagarajan, T., Banerjee, S., Johnson, D. L., Dalton, J. T., Sullivan, R. D., Miller, D. D., Bridges, D., Narayanan, R. Pharmacologic activation of estrogen receptor ß increases mitochondrial function, energy expenditure, and brown adipose tissue.

Diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following H7N9 influenza challenge in mice.

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8(+) cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K(b)PB(1703, D(b)PA(224), and D(b)NP(366) epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.

Seasonal influenza vaccination is the strongest correlate of cross-reactive antibody responses in migratory bird handlers.

UNLABELLED: Avian species are reservoirs of influenza A viruses and could harbor viruses with significant pandemic potential. We examined the antibody and cellular immune responses to influenza A viruses in field or laboratory workers with a spectrum of occupational exposure to avian species for evidence of zoonotic infections. We measured the seroprevalence and T cell responses among 95 individuals with various types and degrees of prior field or laboratory occupational exposure to wild North American avian species using whole blood samples collected in 2010. Plasma samples were tested using endpoint enzyme-linked immunosorbent assay (ELISA) and hemagglutination (HA) inhibition (HAI) assays to subtypes H3, H4, H5, H6, H7, H8, and H12 proteins. Detectable antibodies were found against influenza HA antigens in 77% of individuals, while 65% of individuals tested had measurable T cell responses (gamma interferon [IFN-γ] enzyme-linked immunosorbent spot assay [ELISPOT]) to multiple HA antigens of avian origin. To begin defining the observed antibody specificities, Spearman rank correlation analysis showed that ELISA responses, which measure both head- and stalk-binding antibodies, do not predict HAI reactivities, which measure primarily head-binding antibodies. This result suggests that ELISA titers can report cross-reactivity based on the levels of non-head-binding responses. However, the strongest positive correlate of HA-specific ELISA antibody titers was receipt of seasonal influenza virus vaccination. Occupational exposure was largely uncorrelated with serological measures, with the exception of individuals exposed to poultry, who had higher levels of H7-specific antibodies than non-poultry-exposed individuals. While the cohort had antibody and T cell reactivity to a broad range of influenza viruses, only occupational exposure to poultry was associated with a significant difference in antibody levels to a specific subtype (H7). There was no evidence that T cell assays provided greater specificity for the detection of zoonotic infection. However, influenza vaccination appears to promote cross-reactive antibodies and may provide enhanced protection to novel influenza viruses. IMPORTANCE: Annual vaccinations are necessary to ameliorate influenza disease due to drifted viral variants that emerge in the population. Major shifts in the antigenicity of influenza viruses can result in immunologically distinct viruses that can cause more severe disease in humans. Historically, genetic reassortment between avian, swine, or human influenza viruses has caused influenza pandemics in humans several times in the last century. Therefore, it is important to design vaccines to elicit broad protective responses to influenza infections. Because avian influenza viruses have an important role in emerging infections, we tested whether occupational exposure to birds can elicit immune responses to avian influenza viruses in humans. Instead of a specific occupational exposure, the strongest association of enhanced cross-reactive antibody responses was receipt of seasonal influenza vaccination. Therefore, individuals with preexisting immune responses to seasonal human influenza viruses have substantial cross-reactive antibody and T cell responses that may lead to enhanced protection to novel influenza viruses.

Distinct epigenetic signatures delineate transcriptional programs during virus-specific CD8(+) T cell differentiation.

The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methylation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene transcription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate transcriptional regulation of CTL genes with related functions is achieved via distinct epigenetic mechanisms.

Aging enhances the production of reactive oxygen species and bactericidal activity in peritoneal macrophages by upregulating classical activation pathways.

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.

Proteomic investigation of the time course responses of RAW 264.7 macrophages to infection with Salmonella enterica.

To investigate the extent to which macrophages respond to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serotype Typhimurium and analyzed macrophage proteins at various time points following infection by using a global proteomic approach. A total of 1,006 macrophage and 115 Salmonella proteins were identified with high confidence. Most of the Salmonella proteins were observed in the late stage of the infection time course, which is consistent with the fact that the bacterial cells proliferate inside RAW 264.7 macrophages. The peptide abundances of most of the identified macrophage proteins remained relatively constant over the time course of infection. Compared to those of the control, the peptide abundances of 244 macrophage proteins (i.e., 24% of the total identified macrophage proteins) changed significantly after infection. The functions of these Salmonella-affected macrophage proteins were diverse, including production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase), production of prostaglandin H(2) (i.e., cyclooxygenase 2), and regulation of intracellular traffic (e.g., sorting nexin 5 [SNX5], SNX6, and SNX9). Diverse functions of the Salmonella-affected macrophage proteins demonstrate a global macrophage response to Salmonella infection. Western blot analysis not only confirmed the proteomic results for a selected set of proteins but also revealed that (i) the protein abundance of mitochondrial superoxide dismutase increased following macrophage infection, indicating an infection-induced oxidative stress in mitochondria, and (ii) in contrast to infection of macrophages by wild-type Salmonella, infection by the sopB deletion mutant had no negative impact on the abundance of SNX6, suggesting a role for SopB in regulating the abundance of SNX6.

Calmodulin mediates DNA repair pathways involving H2AX in response to low-dose radiation exposure of RAW 264.7 macrophages.

Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance tumor metastasis. In this respect, we have identified a dose-dependent increase in the abundance (i.e., expression level) of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. At low doses of irradiation there are minimal changes in the abundance of other cellular proteins detected using mass spectrometry, indicating that increases in CaM levels are part of a specific radiation-dependent cellular response. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose radiation exposures. Following macrophage irradiation, increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX foci, associated with DNA repair, with no change in the extent of double-stranded DNA damage. In comparison, when nuclear factor kappaB (NFkappaB)-dependent pathways are inhibited, through the expression of a dominant-negative IkappaB construct, there is no significant increase in phosphorylated histone H2AX foci upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX-mediated DNA repair pathways is not the result of nonspecific NFkappaB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation by enhancing macrophage radioresistance through processes that include the up-regulation of DNA repair pathways involving histone H2AX phosphorylation.

Loss of the calmodulin-dependent inhibition of the RyR1 calcium release channel upon oxidation of methionines in calmodulin.

The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of [3H]ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, in which calcium-activated CaM acts to enhance the calcium sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially weaken the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not significantly affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as part of a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress observed during biological aging. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response that enhances the duration of calcium transients to promote enhanced contractility.

Identification of a denitrase activity against calmodulin in activated macrophages using high-field liquid chromatography--FTICR mass spectrometry.

We have identified a denitrase activity in macrophages that is upregulated following macrophage activation, which is shown by mass spectrometry to recognize nitrotyrosines in the calcium signaling protein calmodulin (CaM). The denitrase activity converts nitrotyrosines to their native tyrosine structure without the formation of any aminotyrosine. Comparable extents of methionine sulfoxide reduction are also observed that are catalyzed by endogenous methionine sulfoxide reductases. Competing with repair processes, oxidized CaM is a substrate for a peptidase activity that results in the selective cleavage of the C-terminal lysine (i.e., Lys148) that is expected to diminish CaM function. Thus, competing repair and peptidase activities define the abundances and functionality of CaM in modulating cellular metabolism in response to oxidative stress, where the presence of the truncated CaM species provides a useful biomarker for the transient appearance of oxidized CaM.

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase.

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.

Increases in calmodulin abundance and stabilization of activated inducible nitric oxide synthase mediate bacterial killing in RAW 264.7 macrophages.

The rapid activation of macrophages in response to bacterial antigens is central to the innate immune system that permits the recognition and killing of pathogens to limit infection. To understand regulatory mechanisms underlying macrophage activation, we have investigated changes in the abundance of calmodulin (CaM) and iNOS in response to the bacterial cell wall component lipopolysaccharide (LPS) using RAW 264.7 macrophages. Critical to these measurements was the ability to differentiate free iNOS from the CaM-bound (active) form of iNOS associated with nitric oxide generation. We observe a rapid 2-fold increase in CaM abundance during the first 30 min that is blocked by inhibition of either NFkappaB nuclear translocation or protein synthesis. A similar 2-fold increase in the abundance of the complex between CaM and iNOS is observed with the same time dependence. In contrast, there are no detectable increases in the CaM-free (i.e., inactive) form of iNOS within the first 2 h; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected macrophages results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following infection by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundance function in the stabilization and activation of iNOS on the rapid time scale associated with macrophage activation and bacterial killing. These results explain how CaM and iNOS coordinately function to form a stable complex that is part of a rapid host response that functions within the first 30 min following bacterial infection to upregulate the innate immune system involving macrophage activation.

Proteomic analysis of Salmonella enterica serovar typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhimurium inside macrophages.

To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.