Pyruvate affects inflammatory responses of macrophages during influenza A virus infection.

Pyruvate is the end product of glycolysis and transported into the mitochondria for use in the tricarboxylic acid (TCA) cycle. It is also a common additive in cell culture media. We discovered that inclusion of sodium pyruvate in culture media during infection of mouse bone marrow derived macrophages with influenza A virus impaired cytokine production (IL-6, IL-1ß, and TNF-α). Sodium pyruvate did not inhibit viral RNA replication. Instead, the addition of sodium pyruvate alters cellular metabolism and diminished mitochondrial reactive oxygen species (ROS) production and lowered immune signaling. Overall, sodium pyruvate affects the immune response produced by macrophages but does not inhibit virus replication.

Deficiency of the NOD-Like Receptor NLRC5 Results in Decreased CD8+ T Cell Function and Impaired Viral Clearance.

Pathogen recognition receptors are vital components of the immune system. Engagement of these receptors is important not only for instigation of innate immune responses to invading pathogens but also for initiating the adaptive immune response. Members of the NOD-like receptor (NLR) family of pathogen recognition receptors have important roles in orchestrating this response. The NLR family member NLRC5 regulates major histocompatibility complex class I (MHC-I) expression during various types of infections, but its role in immunity to influenza A virus (IAV) is not well studied. Here we show that Nlrc5-/- mice exhibit an altered CD8+ T cell response during IAV infection compared to that of wild-type (WT) mice. Nlrc5-/- mice have decreased MHC-I expression on hematopoietic cells and fewer CD8+ T cells prior to infection. NLRC5 deficiency does not affect the generation of antigen-specific CD8+ T cells following IAV infection; however, a change in epitope dominance is observed in Nlrc5-/- mice. Moreover, IAV-specific CD8+ T cells from Nlrc5-/- mice have impaired effector functions. This change in the adaptive immune response is associated with impaired viral clearance in Nlrc5-/- mice. Collectively, our results demonstrate an important role for NLRC5 in regulation of antiviral immune responses and viral clearance during IAV infection.IMPORTANCE The NOD-like receptor family member NLRC5 is known to regulate expression of MHC-I as well as other genes required for antigen processing. In addition, NLRC5 also regulates various immune signaling pathways. In this study, we investigated the role of NLRC5 during influenza virus infection and found a major role for NLRC5 in restricting virus replication and promoting viral clearance. The observed increases in viral titers in NLRC5-deficient mice correlated with impaired effector CD8+ T cell responses. Although NLRC5-deficient mice were defective at clearing the virus, they did not show an increase in morbidity or mortality following influenza virus infection because of other compensatory immune mechanisms. Therefore, our study highlights how NLRC5 regulates multiple immune effector mechanisms to promote the host defense during influenza virus infection.

Integrating Inflammasome Signaling in Sexually Transmitted Infections.

Inflammasomes are cytosolic multiprotein platforms with pivotal roles in infectious diseases. Activation of inflammasomes results in proinflammatory cytokine signaling and pyroptosis. Sexually transmitted infections (STIs) are a major health problem worldwide, yet few studies have probed the impact of inflammasome signaling during these infections. Due to the dearth of appropriate infection models, our current understanding of inflammasomes in STIs is mostly drawn from results obtained in vitro, from distant infection sites, or from related microbial strains that are not sexually transmitted. Understanding how inflammasomes influence the outcome of STIs may lead to the development of novel and effective strategies to control disease and prevent transmission. Here we discuss and highlight the recent progress in this field.

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection.

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell.

Inflammasome control of viral infection.

The inflammasome is a caspase-1 containing complex that activates the proinflammatory cytokines IL-1ß and IL-18 and results in the proinflammatory cell death known as pyroptosis. Numerous recent publications have highlighted the importance of inflammasome activation in the control of virus infection. Inflammasome activation during viral infection is dependent on a variety of upstream receptors including the NOD-like receptor, RIG-I-like receptor and AIM2-like receptor families. Various receptors also function in inflammasome activation in different cellular compartments, including the cytoplasm and the nucleus. The effectiveness of inflammasomes at suppressing virus replication is highlighted by the prevalence and diversity of virus encoded inflammasome inhibitors. Also, the host has a myriad of regulatory mechanisms in place to prevent unwanted inflammasome activation and overt inflammation. Finally, recent reports begin to suggest that inflammasome activation and inflammasome modulation may have important clinical applications. Herein, we highlight recent advances and discuss potential future directions toward understanding the role of inflammasomes during virus infection.

The ketone metabolite ß-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease.

The ketone bodies ß-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein-coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1ß and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1ß secretion in mouse models of NLRP3-mediated diseases such as Muckle-Wells syndrome, familial cold autoinflammatory syndrome and urate crystal-induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Reactive oxygen species regulate caspase-11 expression and activation of the non-canonical NLRP3 inflammasome during enteric pathogen infection.

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3- and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway.

Inhibition of influenza A H3N8 virus infections in mice by morpholino oligomers.

New methods to combat influenza A virus (FLUAV) in humans and animals are needed. The H3N8 subtype virus was the cause of the pandemic of 1890 and has recently undergone cross-species transmission from horses to dogs in the USA. In 2007 H3N8 spread to Australia, a continent previously devoid of equine influenza. Here, we show that antisense-peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), delivered by intranasal administration, are able to inhibit the replication of FLUAV A/Eq/Miami/1/63 (H3N8) in mice by over 95% compared to controls. Monitoring of body weight and immune cell infiltrates in the lungs of noninfected mice indicated that PPMO treatment was not toxic at a concentration shown to be effectively antiviral in vivo. In addition, we detected a naturally occurring mutation within the PPMO target site of a viral gene that may be the cause of resistance to one of the two antisense PPMO sequences tested. These data indicate that PPMOs targeting highly conserved regions of FLUAV are promising novel therapeutic candidates.