Chenopodium quinoa to Modulate Innate Myeloid Cells in the Induction of Obesity

Complex interactions between innate and adaptive immune effectors are an important component in the induction of obesity. Particularly, different subsets of myeloid cells play key roles in metabolic liver diseases and, therefore, are promising targets for intervention strategies. Chenopodium quinoa seeds constitute a good source of immunonutritional compounds, which help prevent high-fat, diet-enhanced innate immune signaling via TLR4/MyD88 that boosts inflammation. Herein, two metabolic mouse models—wild type (WT) and tributyltin treated (TBT)—were used to examine the effects associated with non-alcoholic fatty liver disease (NAFLD); mice were fed with a high-fat diet (HFD) and administered with wheat or C. quinoa bread. Variations in myeloid cells were obtained from a hemogram analysis, and rt-qPCR (mRNA) served to evaluate macrophage markers (i.e., CD68/CD206 ratio) as well as liver inflammation (i.e., Lyve-1) to gain insights into their selective functional differentiation into metabolically injured livers. Only administration of C. quinoa bread prevented alterations in the liver/body weight ratio either in WT animals or those treated with TBT. These effects were associated with significantly increased variations in the peripheral myeloid cell population. Hepatic mRNA markers revealed that C. quinoa enables a selective functional differentiation and function of intrahepatic monocyte-derived macrophages preserving tissue integrity and function.

mtROS Induced via TLR-2-SOCE Signaling Plays Proapoptotic and Bactericidal Role in Mycobacterium fortuitum-Infected Head Kidney Macrophages of Clarias gariepinus

The mechanisms underlying Mycobacterium fortuitum-induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2–endoplasmic reticulum (ER) stress–store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum-infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP, STIM1, and Orai1, which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum-induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.

Lactococcus lactis sb. cremoris orchestrates signal events in the gut epithelium via TLR2 to promote tissue restitution

The use of beneficial bacteria to promote gastrointestinal heath is widely practiced, however, the mechanisms whereby many of these microbes elicit their beneficial effects remain elusive. Previously, we conducted a screen for the discovery of novel beneficial microbes and identified the potent cytoprotective effects of a strain of Lactococcus lactis subsp. cremoris. Here, we show that dietary supplementation with L. lactis subsp. cremoris induced transcript enrichment of a set of genes within the colon whose functions are associated with host cell and microbe interactions. Specifically, L. lactis subsp. cremoris induced the expression of tlr2, which we show was required for L. lactis subsp. cremoris to elicit its beneficial effects on the intestine. L. lactis subsp. cremoris did not confer beneficial effects in mice deficient in TLR-2, or deficient in its adaptor protein Myd88 in chronic gut injury models. In addition to cytoprotection, culture supernatant from L. lactis subsp. cremoris accelerated epithelial migration in a cultured epithelial cell scratch wound assay; and effect that was abrogated by a TLR-2 antagonist. Furthermore, L. lactis subsp. cremoris accelerated epithelial tissue restitution following the infliction of a colonic wound biopsy in a TLR-2 and Myd88-dependent manner. Within colonic wounds, L. lactis subsp. cremoris induced the activation of signaling pathways that function in tissue restitution following injury, including the ERK signaling pathway, and of focal adhesion complex (FAC) proteins. Together, these data demonstrate that L. lactis subsp. cremoris signals via the TLR2/MyD88-axis to confer cytoprotection and accelarated tissue restituion in the gut epithelium. These data point to evolving adaptations where beneficial gut microbes moduate innate immune signaling to excert positive influnces on host physiology.

Lyssaviruses and the Fatal Encephalitic Disease Rabies

Lyssaviruses cause the disease rabies, which is a fatal encephalitic disease resulting in approximately 59,000 human deaths annually. The prototype species, rabies lyssavirus, is the most prevalent of all lyssaviruses and poses the greatest public health threat. In Africa, six confirmed and one putative species of lyssavirus have been identified. Rabies lyssavirus remains endemic throughout mainland Africa, where the domestic dog is the primary reservoir – resulting in the highest per capita death rate from rabies globally. Rabies is typically transmitted through the injection of virus-laden saliva through a bite or scratch from an infected animal. Due to the inhibition of specific immune responses by multifunctional viral proteins, the virus usually replicates at low levels in the muscle tissue and subsequently enters the peripheral nervous system at the neuromuscular junction. Pathogenic rabies lyssavirus strains inhibit innate immune signaling and induce cellular apoptosis as the virus progresses to the central nervous system and brain using viral protein facilitated retrograde axonal transport. Rabies manifests in two different forms - the encephalitic and the paralytic form - with differing clinical manifestations and survival times. Disease symptoms are thought to be due mitochondrial dysfunction, rather than neuronal apoptosis. While much is known about rabies, there remain many gaps in knowledge about the neuropathology of the disease. It should be emphasized however, that rabies is vaccine preventable and dog-mediated human rabies has been eliminated in various countries. The global elimination of dog-mediated human rabies in the foreseeable future is therefore an entirely feasible goal.

Combating Age-Associated Immune Decline Using Kynurenine Pathway Interventions

Select kynurenine pathway interventions extend lifespan in invertebrate models and are of interest in treating age-associated diseases. Kynurenine pathway activity is responsive to inflammatory signaling, and we are evaluating the potential for these interventions to increase pathogen resistance and curtail age-associated immune decline in Caenorhabditis elegans and mammals. The kynurenine pathway facilitates the catabolism of tryptophan to nicotinamide adenine dinucleotide (NAD). Our lab has found that supplementing the kynurenine metabolite 3-hydroxyanthranilic acid (3HAA) or inhibiting the enzyme 3HAA dioxygenase (HAAO) extends lifespan in C. elegans. 3HAA has demonstrated pro/anti-inflammatory properties in mammals, suggesting a potential role in immune function. C. elegans have a primitive immune system that lacks an adaptive element, but it recapitulates aspects of innate immune signaling and pathogen response. I hypothesize kynurenine pathway interventions that impact C. elegans’ lifespan similarly improve pathogen resistance and immunity. Interventions within the kynurenine pathway are capable of differentially impacting pathogenesis and lifespan of C. elegans challenged with Psuedomonas aeruginosa. C. elegans subjected to select lifespan-extending kynurenine pathway interventions fared better when challenged with P. aeruginosa at older ages. Additionally, fluorescent infection tracking has displayed decreased infection rates in worms with elevated 3HAA. Our data suggests pro-immune activity is facilitated by 3HAA acting downstream of the dbl-1 pathway in addition to directly inhibiting bacterial growth. Our goal is to discover the mechanism(s) through which the kynurenine pathway interacts with immune function in animals and identify potential targets for clinical therapy in aging populations.

Regulation of antiviral innate immune signaling and viral evasion following viral genome sensing

A harmonized balance between positive and negative regulation of pattern recognition receptor (PRR)-initiated immune responses is required to achieve the most favorable outcome for the host. This balance is crucial because it must not only ensure activation of the first line of defense against viral infection but also prevent inappropriate immune activation, which results in autoimmune diseases. Recent studies have shown how signal transduction pathways initiated by PRRs are positively and negatively regulated by diverse modulators to maintain host immune homeostasis. However, viruses have developed strategies to subvert the host antiviral response and establish infection. Viruses have evolved numerous genes encoding immunomodulatory proteins that antagonize the host immune system. This review focuses on the current state of knowledge regarding key host factors that regulate innate immune signaling molecules upon viral infection and discusses evidence showing how specific viral proteins counteract antiviral responses via immunomodulatory strategies.

Insights into the SARS-CoV-2-Mediated Alteration in the Stress Granule Protein Regulatory Networks in Humans

The rapidly and constantly evolving coronavirus, SARS-CoV-2, imposes a great threat to human health causing severe lung disease and significant mortality. Cytoplasmic stress granules (SGs) exert anti-viral activities due to their involvement in translation inhibition and innate immune signaling. SARS-CoV-2 sequesters important SG nucleator proteins and impairs SG formation, thus evading the host response for efficient viral replication. However, the significance of SGs in COVID-19 infection remains elusive. In this study, we utilize a protein-protein interaction network approach to systematically dissect the crosstalk of human post-translational regulatory networks governed by SG proteins due to SARS-CoV-2 infection. We uncovered that 116 human SG proteins directly interact with SARS-CoV-2 proteins and are involved in 430 different brain disorders including COVID-19. Further, we performed gene set enrichment analysis to identify the drugs against three important key SG proteins (DYNC1H1, DCTN1, and LMNA) and also looked for potential microRNAs (miRNAs) targeting these proteins. We identified bexarotene as a potential drug molecule and miRNAs, hsa-miR-615-3p, hsa-miR-221-3p, and hsa-miR-124-3p as potential candidates for the treatment of COVID-19 and associated manifestations.

Combination of Ras Modulator and Azacitidine Impacts Innate Immune Signaling Pathway in MDS-L Cell Line

Background: Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis, uni/multi-lineage dysplasia and a high tendency to transform into acute myeloid leukemia. Aberrant chromosomal and genetic lesions contribute to MDS pathogenesis which has been associated with chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al. Blood, 2018). Dysfunction of Toll like receptors (TLR) and downstream effectors has been associated with the loss of progenitor function and differentiation of bone marrow (BM) cells in MDS patients. Azacitidine (AZA), a hypomethylating agent (HMA), is the mainstay of therapy for patients with higher-risk MDS (Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017) and carries an overall response rate (ORR) of 50% in patients with significant effects on hematopoiesis, ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence with survival benefits (Silverman LR, et al., Leukemia, 1993). The response to AZA is not durable and all patients relapse with worsening bone marrow failure. The paradigm of MDS therapy is now shifting to combinatorial drug treatment to overcome single-agent HMA resistance in higher-risk MDS patients. Rigosertib (RIGO), a Ras mimetic which had been shown to interfere with the Ras-Raf binding domain, has limited single-agent activity (ORR 15%) and failed to provide a survival benefit compared to standard of care in MDS patients failing an HMA. RIGO combined with AZA produced an ORR of 90% in HMA naïve patients and 54% in patients who failed HMA (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The mechanism is still unclear. Method: We therefore investigated the pathways that are perturbed by AZA and RIGO monotherapy and in combination (RIGO-AZA). We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with RIGO, AZA, and RIGO-AZA for 48 hrs and further analyzed by qPCR and western blot. Result: We found an increase in H3K9ac protein expression with RIGO and RIGO-AZA; AZA and RIGO alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RIGO-AZA (Figure A). Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RIGO and RIGO-AZA combination (Figure A). Furthermore, we studied the expression of bacterial sensing TLRs (1, 2 and 6), viral sensing endosomal TLRs (3 and 9), and cytosolic viral particle sensing receptors like Retinoic acid inducible gene (RIG)-I, Melanoma differentiation-associated protein 5 (MDA5) and Stimulator of interferon genes (STING); their intermediate adaptor molecules Myeloid differentiation factor 88 (MYD88) (for all TLRs except TLR3), mitochondrial antiviral signaling (MAVS) gene (for RIG-I and MDA5); and interferon regulatory factor (IRF)-3 and -7 by qPCR. We observed that AZA, RIGO, and RIGO-AZA significantly inhibit the expression of TLR1, 2 and 6 (Figure B). However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RIGO and RIGO-AZA (Figure C-E). Conclusion: RIGO has effects on innate immune signaling and histone modification of both activator and repressor marks. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, and if these mechanisms contribute to the improvement in hematopoiesis in MDS patients. Figure 1 Figure 1. Disclosures Navada: Janssen Pharmaceuticals, Inc.: Current Employment. Reddy: Onconova Therapeutics, Inc.: Current equity holder in publicly-traded company.

Cigarette Smoking Exacerbates Flu-Induced Thrombo-Inflammation

Rationale: Epidemiological evidence suggests that prior exposure to cigarette smoke (CS) or habitual smoking increases the risk of influenza A virus (IAV)-triggered respiratory failure (severe flu). Although emerging evidence supports the role of thrombo-inflammation in the development of CS and IAV-triggered lung injury, the innate immune mechanism that contributes to this morbidity remains poorly understood. Materials and methods: We have developed a two-hit model of CS-induced severe flu in mice. Mice were exposed to four weeks of room air (air) or CS followed by intra-nasal administration of A/PR/8/34 (H1N1) IAV. The body weight was measured every day for two weeks after IAV administration followed by assessment of lung injury at day 7 and 14. Lungs were harvested for histological assessment of lung injury and estimation of viral titer by RT-PCR. Quantitative fluorescence intravital lung microscopy (qFILM) was conducted at 2-, 3- and 4-days post IAV-infection to visualize dynamics of neutrophil and platelet recruitment in the lung of mice IV administered with fluorescent dextran, anti-Ly6G Ab and anti-CD49Ab. Results: Mice exposed to CS+IAV manifested significantly more weight loss, lung injury, lung congestion and alveolar hemorrhage compared to mice administered room-air+IAV. QFILM revealed that severity of lung injury was associated with significantly more entrapment of neutrophil-platelet aggregates within the pulmonary microcirculation and infiltration into the air spaces of CS+IAV than room-air+IAV administered mice. Conclusion: These initial results suggest that CS primes innate immune signaling in neutrophils and platelets to promote their recruitment in the lung following flu. Currently, studies are underway to identify innate immune pathways in neutrophils and platelets that drive this severe thrombo-inflammatory response. Disclosures Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Slc39a2-Mediated Zinc Homeostasis Modulates Innate Immune Signaling in Phenylephrine-Induced Cardiomyocyte Hypertrophy

Zinc dyshomeostasis has been involved in the pathogenesis of cardiac hypertrophy; however, the dynamic regulation of intracellular zinc and its downstream signaling in cardiac hypertrophy remain largely unknown. Using Zincpyr1 staining, we found a significant decrease of intracellular Zinc concentration in phenylephrine (PE)-induced hypertrophy of neonatal rat ventricular myocytes (NRVMs). We then screened SLC39 family members responsible for zinc uptake and identified Slc39a2 as the only one altered by PE treatment. Slc39a2 knockdown in NRVMs reduced the intracellular Zinc level, and exacerbated the hypertrophic responses to PE treatment. In contrast, adenovirus-mediated Slc39a2 overexpression enhanced zinc uptake and suppressed PE-induced Nppb expression. RNA sequencing analysis showed a pro-hypertrophic transcriptome reprogramming after Slc39a2 knockdown. Interestingly, the innate immune signaling pathways, including NOD signaling, TOLL-like receptor, NFκB, and IRFs, were remarkably enriched in the Slc39a2-regulated genes. Slc39a2 deficiency enhanced the phosphorylation of P65 NFκB and STAT3, and reduced the expression of IκBα. Finally, the expression of IRF7 was significantly increased by Slc39a2 knockdown, which was in turn suppressed by IRF7 knockdown. Our data demonstrate that zinc homeostasis mediated by a Slc39a2/IRF7 regulatory circuit contributes to the alteration of innate immune signaling in cardiomyocyte hypertrophy.