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1. Fusogenic porous silicon nanoparticles as a broad-spectrum immunotherapy against bacterial infections

   https://doi.org/10.1039/D0NH00624F  

   Kim, Byungji ; Yang, Qinglin ; Chan, Leslie W. ; Bhatia, Sangeeta N. ; Ruoslahti, Erkki ; Sailor, Michael J. ( April 2021 , Nanoscale Horizons)

   null (Ed.)

   Bacterial infections are re-emerging as substantial threats to global health due to the limited selection of antibiotics that are capable of overcoming antibiotic-resistant strains. By deterring such mutations whilst minimizing the need to develop new pathogen-specific antibiotics, immunotherapy offers a broad-spectrum therapeutic solution against bacterial infections. In particular, pathology resulting from excessive immune response ( i.e. fibrosis, necrosis, exudation, breath impediment) contributes significantly to negative disease outcome. Herein, we present a nanoparticle that is targeted to activated macrophages and loaded with siRNA against the Irf5 gene. This formulation is able to induce >80% gene silencing in activated macrophages in vivo , and it inhibits the excessive inflammatory response, generating a significantly improved therapeutic outcome in mouse models of bacterial infection. The versatility of the approach is demonstrated using mice with antibiotic-resistant Gram-positive (methicillin-resistant Staphylococcus aureus ) and Gram-negative ( Pseudomonas aeruginosa ) muscle and lung infections, respectively. Effective depletion of the Irf5 gene in macrophages is found to significantly improve the therapeutic outcome of infected mice, regardless of the bacteria strain and type. 

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2. Inflammasome Assays In Vitro and in Mouse Models

   https://doi.org/10.1002/cpim.107  

   Guo, Haitao ; Ting, Jenny P. ‐Y. ( October 2020 , Current Protocols in Immunology)

   This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll‐like receptor agonists and TNF‐α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation‐mediated maturation of interleukin (IL)‐1β and IL‐18 and pyroptosis. Since the well‐established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis‐associated speck‐like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti‐ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL‐1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high‐dose LPS challenge in a sepsis model. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Priming and activation of inflammasomes in mouse macrophages

   Basic Protocol 2: Activation of human NLRP1 inflammasome by DPP8/9 inhibitor talabostat

   Basic Protocol 3: Purification and detection of ASC pyroptosome

   Alternate Protocol 1: Detection of ASC speck by immunofluorescence staining

   Basic Protocol 4: Activation of canonical NLRP3 inflammasome in mice by intraperitoneal delivery of MSU crystals

   Alternate Protocol 2: Activation of noncanonical NLRP3 inflammasome in mice by intraperitoneal delivery of LPS

    

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3. Frontline Science: Escherichia coli use LPS as decoy to impair neutrophil chemotaxis and defeat antimicrobial host defense

   https://doi.org/10.1002/JLB.4HI0319-109R  

   Kondo, Yutaka ; Ledderose, Carola ; Slubowski, Christian J. ; Fakhari, Mahtab ; Sumi, Yuka ; Sueyoshi, Koichiro ; Bezler, Ann-Katrin ; Aytan, Dilan ; Arbab, Mona ; Junger, Wolfgang G. ( August 2019 , Journal of Leukocyte Biology)

   Bacterial infections and sepsis are leading causes of morbidity and mortality in critically ill patients. Currently, there are no effective treatments available to improve clinical outcome in sepsis. Here, we elucidated a mechanism by which Escherichia coli (E. coli) bacteria impair neutrophil (PMN) chemotaxis and we studied whether this mechanism can be therapeutically targeted to improve chemotaxis and antimicrobial host defense. PMNs detect bacteria with formyl peptide receptors (FPR). FPR stimulation triggers mitochondrial ATP production and release. Autocrine stimulation of purinergic receptors exerts excitatory and inhibitory downstream signals that induce cell polarization and cell shape changes needed for chemotaxis. Here we show that the bacterial cell wall product LPS dose-dependently impairs PMN chemotaxis. Exposure of human PMNs to LPS triggered excessive mitochondrial ATP production and disorganized intracellular trafficking of mitochondria, resulting in global ATP release that disrupted purinergic signaling, cell polarization, and chemotaxis. In mice infected i.p. with E. coli, LPS treatment increased the spread of bacteria at the infection site and throughout the systemic circulation. Removal of excessive systemic ATP with apyrase improved chemotaxis of LPS-treated human PMNs in vitro and enhanced the clearance of E. coli in infected and LPS-treated mice. We conclude that systemic ATP accumulation in response to LPS is a potential therapeutic target to restore PMN chemotaxis and to boost the antimicrobial host immune defense in sepsis.

    

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4. The Role of Lipopolysaccharide-Induced Extracellular Vesicles in Cardiac Cell Death

   https://doi.org/10.3390/biology8040069  

   Bell, Courtnee’ R. ; Jones, Leandra B. ; Crenshaw, Brennetta J. ; Kumar, Sanjay ; Rowe, Glenn C. ; Sims, Brian ; Javan, Gulnaz T. ; Matthews, Qiana L. ( December 2019 , Biology)

   Exosomes play a crucial role in the progression of infectious diseases, as exosome release and biogenesis are affected by external factors, such as pathogenic infections. Pyrogens may aide in the progression of diseases by triggering inflammation, endothelial cell injury, and arterial plaque rupture, all of which can lead to acute coronary disease, resulting in cardiac tissue death and the onset of a cardiac event (CE). To better understand the effects of Gram-negative bacterial infections on exosome composition and biogenesis, we examined exosome characteristics after treatment of AC16 human cardiomyocytes with lipopolysaccharide (LPS), which served as a model system for Gram-negative bacterial infection. Using increasing doses (0, 0.1, 1, or 10 µg) of LPS, we showed that treatment with LPS substantially altered the composition of AC16-derived exosomes. Both the relative size and the quantity (particles/mL) of exosomes were decreased significantly at all tested concentrations of LPS treatment compared to the untreated group. In addition, LPS administration reduced the expression of exosomal proteins that are related to exosomal biogenesis. Conversely, we observed an increase in immunomodulators present after LPS administration. This evaluation of the impact of LPS on cardiac cell death and exosome composition will yield new insight into the importance of exosomes in a variety of physiological and pathological processes as it relates to disease progression, diagnosis, and treatment. 

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5. The Route of Infection Influences the Contribution of Key Immunity Genes to Antibacterial Defense in Anopheles gambiae

   https://doi.org/10.1159/000511401  

   Dekmak, Amira San ; Yang, Xiaowei ; Zu Dohna, Heinrich ; Buchon, Nicolas ; Osta, Mike A. ( March 2021 , Journal of Innate Immunity)

   null (Ed.)

   Insect systemic immune responses to bacterial infections have been mainly studied using microinjections, whereby the microbe is directly injected into the hemocoel. While this methodology has been instrumental in defining immune signaling pathways and enzymatic cascades in the hemolymph, it remains unclear whether and to what extent the contribution of systemic immune defenses to host microbial resistance varies if bacteria invade the hemolymph after crossing the midgut epithelium subsequent to an oral infection. Here, we address this question using the pathogenic Serratia marcescens (Sm) DB11 strain to establish systemic infections of the malaria vector Anopheles gambiae, either by septic Sm injections or by midgut crossing after feeding on Sm. Using functional genetic studies by RNAi, we report that the two humoral immune factors, thioester-containing protein 1 and C-type lectin 4, which play key roles in defense against Gram-negative bacterial infections, are essential for defense against systemic Sm infections established through injection, but they become dispensable when Sm infects the hemolymph following oral infection. Similar results were observed for the mosquito Rel2 pathway. Surprisingly, blocking phagocytosis by cytochalasin D treatment did not affect mosquito susceptibility to Sm infections established through either route. Transcriptomic analysis of mosquito midguts and abdomens by RNA-seq revealed that the transcriptional response in these tissues is more pronounced in response to feeding on Sm. Functional classification of differentially expressed transcripts identified metabolic genes as the most represented class in response to both routes of infection, while immune genes were poorly regulated in both routes. We also report that Sm oral infections are associated with significant downregulation of several immune genes belonging to different families, specifically the clip-domain serine protease family. In sum, our findings reveal that the route of infection not only alters the contribution of key immunity genes to host antimicrobial defense but is also associated with different transcriptional responses in midguts and abdomens, possibly reflecting different adaptive strategies of the host. 

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