Microglial activation in Alzheimer's disease: The role of flavonoids and microRNAs.

Alzheimer's disease (AD) is the most common form of senile dementia and is characterized by progressive cognitive impairment and neuronal degeneration. Microglial activation is an important pathologic hallmark of AD. During disease progression, microglial cells switch from an alternative or anti-inflammatory and neuroprotective profile (M2) to a classic or proinflammatory and neurotoxic profile (M1). Phenotypically, M1 microglia is characterized by the activation of inflammatory signaling pathways that cause increased expression of proinflammatory genes, including those coding for cytokines and chemokines. This microglia-mediated neuroinflammation contributes to neuronal cell death. Recent studies in microglial cells have shown that a group of plant-derived compounds, known as flavonoids, possess anti-inflammatory properties and therefore exert a neuroprotective effect through regulating microglia activation. Here, we discuss how flavonoids can promote the switch from an inflammatory M1 phenotype to an anti-inflammatory M2 phenotype in microglia and how this represents a valuable opportunity for the development of novel therapeutic strategies to blunt neuroinflammation and boost neuronal recovery in AD. We also review how certain flavonoids can inhibit neuroinflammation through their action on the expression of microglia-specific microRNAs (miRNAs), which also constitute a key therapeutic approach in different neuropathologies involving an inflammatory component, including AD. Finally, we propose novel targets of microglia-specific miRNAs that may be considered for AD treatment.

PEGylation of cytochrome P450 enhances its biocatalytic performance for pesticide transformation.

Pesticide intoxication is a major public health concern, and unfortunately there is not an effective treatment for severe organophosphorus pesticide intoxication. In this work, a non-immunogenic enzymatic bioconjugate based on cytochrome P450 was assayed for organophosphorus pesticide transformation. Enzyme therapy is an alternative approach to inactivate pesticides in the bloodstream, transforming them into less toxic metabolites. A variant of cytochrome P450 (CYPBM3 F87A) from Bacillus megaterium was chemically modified with polyethylene glycol. The PEGylated enzyme showed enhanced pesticide transformation activity when compared with the unmodified protein. The transformation rates were higher than those obtained with the unmodified enzyme for all six pesticides transformed. The specific activity of PEGylated preparation for parathion and dichlorophen was up to 9-times higher than these obtained with the unmodified enzyme. In addition, the modified CYP (CYP-PEG) remained active at extremely high pHs, maintaining 90% of its maximal activity at pH 11, as opposed to the unmodified CYP that retained less than 20% of its maximal activity at that pH. In addition, the bioconjugate showed good catalytic activity in blood serum and innocuousness on immune cells. The potential use of PEGylated CYP as a detoxification strategy for pesticide poisoning is demonstrated and discussed.

Erythrocyte sialoglycoproteins engage Siglec-9 on neutrophils to suppress activation.

Healthy blood neutrophils are functionally quiescent in the bloodstream, have a short lifespan, and exit the circulation to carry out innate immune functions, or undergo rapid apoptosis and macrophage-mediated clearance to mitigate host tissue damage. Limitation of unnecessary intravascular neutrophil activation is also important to prevent serious inflammatory pathologies. Because neutrophils become easily activated after purification, we carried out ex vivo comparisons with neutrophils maintained in whole blood. We found a difference in activation state, with purified neutrophils showing signs of increased reactivity: shedding of l-selectin, CD11b upregulation, increased oxidative burst, and faster progression to apoptosis. We discovered that erythrocytes suppressed neutrophil activation ex vivo and in vitro, including reduced l-selectin shedding, oxidative burst, chemotaxis, neutrophil extracellular trap formation, bacterial killing, and induction of apoptosis. Selective and specific modification of sialic acid side chains on erythrocyte surfaces with mild sodium metaperiodate oxidation followed by aldehyde quenching with 4-methyl-3-thiosemicarbazide reduced neutrophil binding to erythrocytes and restored neutrophil activation. By enzyme-linked immunosorbent assay and immunofluorescence, we found that glycophorin A, the most abundant sialoglycoprotein on erythrocytes, engaged neutrophil Siglec-9, a sialic acid-recognizing receptor known to dampen innate immune cell activation. These studies demonstrate a previously unsuspected role for erythrocytes in suppressing neutrophils ex vivo and in vitro and help explain why neutrophils become easily activated after separation from whole blood. We propose that a sialic acid-based "self-associated molecular pattern" on erythrocytes also helps maintain neutrophil quiescence in the bloodstream. Our findings may be relevant to some prior experimental and clinical studies of neutrophils.

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen.

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.

Host and pathogen hyaluronan signal through human siglec-9 to suppress neutrophil activation.

UNLABELLED: Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism. KEY MESSAGE: HMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.

Positive regulation of TRAF6-dependent innate immune responses by protein phosphatase PP1-γ.

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.

Loss of Siglec-14 reduces the risk of chronic obstructive pulmonary disease exacerbation.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality worldwide. COPD exacerbation, or episodic worsening of symptoms, often results in hospitalization and increased mortality rates. Airway infections by new bacterial strains, such as nontypeable Haemophilus influenzae (NTHi), are a major cause of COPD exacerbation. NTHi express lipooligosaccharides that contain sialic acids, and may interact with Siglec-14, a sialic acid recognition protein on myeloid cells that serves as an activating signal transduction receptor. A null allele polymorphism in SIGLEC14 may attenuate the inflammatory responses to NTHi by eliminating Siglec-14 expression. We asked if the loss of Siglec-14 attenuates the inflammatory response by myeloid cells against NTHi, and if the SIGLEC14-null polymorphism has any effect on COPD exacerbation. We found that NTHi interacts with Siglec-14 to enhance proinflammatory cytokine production in a tissue culture model. Inhibitors of the Syk tyrosine kinase suppress this response. Loss of Siglec-14, due to SIGLEC14-null allele homozygosity, is associated with a reduced risk of COPD exacerbation in a Japanese patient population. Taken together, Siglec-14 and its downstream signaling pathway facilitate the "infection-inflammation-exacerbation" axis of COPD disease progression, and may represent promising targets for therapeutic intervention.

Specific inactivation of two immunomodulatory SIGLEC genes during human evolution.

Sialic acid-recognizing Ig-like lectins (Siglecs) are signaling receptors that modulate immune responses, and are targeted for interactions by certain pathogens. We describe two primate Siglecs that were rendered nonfunctional by single genetic events during hominin evolution after our common ancestor with the chimpanzee. SIGLEC13 was deleted by an Alu-mediated recombination event, and a single base pair deletion disrupted the ORF of SIGLEC17. Siglec-13 is expressed on chimpanzee monocytes, innate immune cells that react to bacteria. The human SIGLEC17P pseudogene mRNA is still expressed at high levels in human natural killer cells, which bridge innate and adaptive immune responses. As both resulting pseudogenes are homozygous in all human populations, we resurrected the originally encoded proteins and examined their functions. Chimpanzee Siglec-13 and the resurrected human Siglec-17 recruit a signaling adapter and bind sialic acids. Expression of either Siglec in innate immune cells alters inflammatory cytokine secretion in response to Toll-like receptor-4 stimulation. Both Siglecs can also be engaged by two potentially lethal sialylated bacterial pathogens of newborns and infants, agents with a potential impact on reproductive fitness. Neanderthal and Denisovan genomes show human-like sequences at both loci, corroborating estimates that the initial pseudogenization events occurred in the common ancestral population of these hominins. Both loci also show limited polymorphic diversity, suggesting selection forces predating the origin of modern humans. Taken together, these data suggest that genetic elimination of Siglec-13 and/or Siglec-17 represents signatures of infectious and/or other inflammatory selective processes contributing to population restrictions during hominin origins.

Cofactors required for TLR7- and TLR9-dependent innate immune responses.

Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.

Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response.

We examined the ability of porins from Salmonella enterica serovar typhi to induce a long-term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long-lasting anti-S. typhi porin sera did not cross-react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal-binding activity to the wild-type S. typhi strain, whereas porin-specific antibody titres measured by enzyme-linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long-lasting antibody response. OmpC and OmpF induced long-lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody-mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant.

Utilidad diagnóstica del lavado broncoalveolar en la neumonía nosocomial asociada con ventilación mecánica en pacientes bajo tratamiento antibiótico / Diagnostic utility of bronchoalveolar lavage in mechanical ventilation-associated nosocomial pneumonia in patients under antibiotic treatment

Bajo los lineamientos de una prueba diagnóstica, determinamos la utilidad del cultivo con cuenta de unidades formadoras de colonias (UFC) en los cultivos de lavado broncoalveolar (LAB) de pacientes con sospecha de neumonía asociada a ventilación mecánica (NAVM) y bajo tratamiento con antibióticos sistémicos. Los cultivos con crecimiento de bacterias en 10 a la cuarta y 10 a la quinta YFC/ml fueron considerados positivos, mientras los cultivos sin crecimiento fueron considerados negativos. Los cultivos con crecimiento de 10 al cubo UFC/ml se consideraron como contaminación. El diagnóstico final de NAVM (estándar de oro) se hizo con criterios de tipo clínico, bacteriológico e histológico. Se estudiaron 12 pacientes con sospecha de NAVM y como grupo control se estudiaron seis pacientes bajo ventilación mecánica, pero sin evidencia de neumonía u otra infección. Todos los pacientes con sospecha de NAVM tuvieron cultivo positivo, mientras que todos los controles tuvieron cultivos negativos. La sensibilidad de la prueba fue de 100 por ciento y la especificidad de 75 por ciento, con un valor predictivo positivo de 88 por ciento y valor predictivo negativo de 100 por ciento. Concluimos que los cultivos con cuenta de UFC de LBA es un método útil para el diagnóstico de la NAVM, aun cuando el paciente se encuentra bajo tratamiento con antibióticos sistémicos