Innate Antimicrobial Immunity Research Articles

Immunoresponsive Gene 1 Facilitates TLR4-agonist-Induced Augmentation of Innate Antimicrobial Immunity.

Treatment with the toll-like receptor (TLR) 4 agonist monophosphoryl lipid A (MPLA) conditions innate immunocytes to respond robustly to subsequent infection, a phenotype termed innate immune memory. Our published studies show that metabolic reprogramming of macrophages is a prominent feature of the memory phenotype. We undertook studies to define the functional contributions of tricarboxylic acid (TCA) cycle reprogramming to innate immune memory. We observed that priming of wild type (WT) mice with MPLA potently facilitated accumulation of the TCA cycle metabolite itaconate at sites of infection and enhanced microbial clearance. Augmentation of itaconate accumulation and microbial clearance was ablated in immuneresponsive gene 1 (Irg1) -deficient mice. We further observed that MPLA potently induces expression of Irg1 and accumulation of itaconate in macrophages. Compared to WT macrophages, the ability of Irg1-deficient macrophages to kill Pseudomonas aeruginosa was impaired. We further observed that itaconate is directly antimicrobial against P. aeruginosa at pH 5, which is characteristic of the phagolysosome, and is facilitated by reactive oxygen species. MPLA-induced augmentation of glycolysis, oxidative phosphorylation and accumulation of the TCA cycle metabolites succinate and malate was decreased in Irg1 KO macrophages compared to WT controls. RNA sequencing revealed suppressed transcription of genes associated with phagolysosome function and increased expression of genes associated with cytokine production and chemotaxis in Irg1 deficient macrophages. This study identifies a contribution of itaconate to MPLA-induced augmentation of innate antimicrobial immunity via facilitation of microbial killing as well as impact on metabolic and transcriptional adaptations.

Characterisation, prevalence and antibiogram of Aeromonas hydrophila in turtles: insights into virulence factors, antimicrobial resistance genes and innate immunity

ABSTRACT Bacterial infection poses a significant threat to the health and survival of turtles, both in their natural habitats and in captivity. Notably, the presence and interactions of bacteria, such as those belonging to the Aeromonas genus, have been identified for their adverse impact on turtle health and ecosystem dynamics. This study investigated the prevalence, antibiotic susceptibility, virulence factors, and antimicrobial resistance genes of Aeromonas hydrophila in Trachemys scripta elegans (Red-eared Slider) and Chelonia mydas (Green Turtle). The study also examined the innate immunity of T. scripta elegans and C. mydas. Buccal and cloacal swabs from 39 turtles were collected and examined alongside 45 water samples to try to isolate and amplify A. hydrophila. Polymerase chain reaction (PCR) amplifications were then sequenced to identify the presence of specific genes associated with virulence, and antimicrobial resistance. The findings revealed that A. hydrophila had a prevalence rate of 35.9% in all examined turtle species, with 14 water samples accounting for 31% of the total samples. Buccal samples from Green and Red-eared turtles showed a higher rate of A. hydrophila isolates than cloacal samples. Sensitivity to norfloxacin (85.71%), gentamycin (71.42%), and ceftriaxone (64.28%) was observed, whereas resistance to amoxicillin (64.28%), ampicillin (57.14%), and rifamycin (57.14%) was apparent. Antibiotic resistance genes, including blaTEM and blaSHV, in A. hydrophila isolates, were present in 42.86% and 28.5% of the samples, respectively. PCR analysis confirmed the genetic identity of A. hydrophila, and five virulence genes were detected: AHCYTOEN (100%), followed by aerolysin (92.8%), alt and act (85.7%), and lipase (78.5%). Genetic sequences of the isolates had a 99% identity with the reference strains in GenBank. Furthermore, the study found the innate immunity of turtles to be robust due to serum lysozyme activity, bactericidal killing assay (BKA), total leukocyte count, heterophil/lymphocyte ratio, and packed cell volume (PCV%), all of which help to protect them from infections. Overall, this research highlights the need for continued monitoring and management of antibiotic resistance in aquatic ecosystems.

African swine fever virus E120R inhibited cGAS‐STING‐mediated IFN‐β and NF‐κB pathways

AbstractAfrican swine fever (ASF) is an acute and severe contagious disease triggered by the African swine fever virus (ASFV), which severely threatens the global swine industry. At present, no safe and efficacious vaccine has been provided to prevent and control this disease. The pathogenesis and immune evasion mechanism of ASFV are still unknown, which seriously hinders the development of safe and effective ASF vaccines. Certain proteins of ASFV involved in immunosuppression helped to evade the host innate immune response. The cGAS‐STING signaling pathway is important to the innate immune system. It induces the production of type I interferons (IFNs) and other cytokines by recognizing cytoplasmic DNA, mediating antimicrobial innate immunity through type I IFN, and nuclear factor‐κB (NF‐κB) pathways. In the present study, E120R, a late‐phase expression protein and a key virulent factor of ASFV inhibited cGAS‐STING mediated promoter activities of IFN‐β and NF‐κB in HEK293T cells. The ectopic expression of E120R down‐regulated IFN‐β pathway by targeting interferon regulatory factor 3 (IRF3) and p65, inhibited the phosphorylation of STING, and further inhibited the phosphorylation of TANK‐binding kinase 1 (TBK1) and IRF3, with no significant effects on p65 phosphorylation. Additionally, E120R also inhibited the NF‐κB pathways by inhibiting the nuclear translocation of p50 and p65, which was mediated by Sendai virus (SeV). Further, the study showed that the 61–80 amino acids sites in the C‐terminal domain of E120R were crucial for these functions. In conclusion, our work preliminarily elucidated a novel mechanism of inhibiting host innate immune response by ASFV E120R, which will provide a new target for the ASFV live gene deletion vaccine development and the theoretical basis for ASFV prevention.

Use of a Tissue-Targeting Recombinant Human Interleukin-22 Fusion Molecule (F-652) for the Treatment of Advanced Refractory Lower GI Acute Gvhd

Background: Steroid-refractory (SR) gastrointestinal acute GVHD (GI aGVHD) is a serious complication after allogeneic hematopoietic transplantation (allo-HCT), and subsequent lines of treatment following steroid failure are often ineffective. The JAK 1/2 inhibitor ruxolitinib is the only FDA-approved treatment for SR aGVHD, and patients for whom ruxolitinib is not effective or not tolerated have limited therapeutic options and high mortality. Interleukin-22 (IL-22) is a tissue-protective IL-10-family cytokine that has been shown to support intestinal mucosa after damage, directly signaling to the intestinal epithelium and promoting its survival, regeneration, and production of innate antimicrobial molecules. F-652, a recombinant human (rh)IL-22 fusion protein composed of an IL-22 dimer and IgG2 Fc fragment, has recently been studied in a phase 2 trial for patients with newly diagnosed lower GI aGVHD, demonstrating a promising day 28 treatment response rate and improved gut microbial health. Given this, we examined F-652 administration in 3 patients with severe SR GI aGVHD. Methods: Three patients with hematologic malignancies underwent MUD allo-HCT with tacrolimus and methotrexate GVHD prophylaxis ( Table). The patients were initially treated for GI aGVHD with methylprednisolone at a dose of 2 mg/kg/day without response. All patients presented with or progressed to stage 4 gut aGVHD and failed several lines of therapy, including ruxolitinib. All patients also had multiple episodes of bacteremia and at least some degree of GVHD-associated GI bleeding. F-652 treatment was initiated under single-patient-use authorization as the 5 th-7 th line of therapy and was administered at a dose of 45 mcg/kg IV weekly. In vitro cultures suggested that rho kinase inhibition did not interfere with trophic effects of IL-22 on intestinal epithelium, so patients were permitted to continue belumosidil upon F-652 initiation. While there was concern that JAK inhibition could compromise the activity of IL-22, continuation of ruxolitinib was considered on a case-by-case basis. C-reactive protein (CRP) is an acute phase reactant induced by IL-22, and circulating CRP levels served as a pharmacodynamic marker of in vivo biologic activity of F-652. Results: Consistent CRP elevations were observed 3 days after F-652 dosing in patients A and C, both of whom demonstrated a reduction in GVHD symptoms following F-652 treatment. These CRP elevations occurred despite concurrent use of belumosidil in Patient A and ruxolitinib in Patient C. CRP levels increased in Patient B after the first two doses, but not the third ( Figure). By day 28 following F-652 initiation, 2-of-3 patients (A and C) achieved a partial response, with decreased stool output and resolution of GI bleeding. Patient A had experienced 21 episodes of bacteremia prior to initiating rhIL-22 therapy but continued to demonstrate a partial response at day 56 and resolutiuon of bacteremias. Further improvement during the continued treatment (12 total doses) allowed for hospital discharge after 14 months of hospitalization. Patient B received 4 failed lines of therapy beyond ruxolitinib and was experiencing GI bleeding necessitating frequent transfusions prior to F-652 initiation. The symptoms persisted following F-652 treatment, and the patient passed at day 149 post-HCT. Patient C continues on active F-652 treatment and remains in a partial response as of this submission (> 50 days after treatment initiation). Overall, F-652 treatment has been well tolerated, and no significant adverse events attributable to the drug were observed. Conclusions: This is the first report of IL-22 therapy in SR aGVHD. Treatment was well tolerated, and 2-of-3 patients achieved a response following rhIL-22 administration after failing multiple previous therapies. Whereas most treatments for GVHD target immune cells and deepen immunosuppression, IL-22 is understood to act directly upon epithelial cells where it can support tissue recovery, improve barrier function, and promote innate antimicrobial immunity. CRP elevations post-treatment suggest that IL-22 administration may be able to exert these effects even with concurrent use of a JAK inhibitor. Our findings support further development of this approach and provide proof-of-concept for the use of tissue-supportive strategies to enhance the recovery of damaged gastrointestinal mucosa in advanced lower GI aGVHD.

Impaired resolution of blood transcriptomes through tuberculosis treatment with diabetes comorbidity.

People with diabetes are more likely to develop tuberculosis (TB) and to have poor TB-treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at diagnosis. It is unknown whether this persists through treatment and contributes to the adverse outcomes. Pulmonary TB patients recruited in South Africa, Indonesia and Romania were classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or TB-only, based on glycated haemoglobin concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood at diagnosis and intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification. Transcriptomic data were analysed by longitudinal mixed-model regression to identify whether genes were differentially expressed between clinical groups through time. Predictive models of TB-treatment response across groups were developed and cross-tested. Gene expression differed between TB and TB-DM patients at diagnosis and was modulated by TB treatment in all clinical groups but to different extents, such that differences remained in TB-DM relative to TB-only throughout. Expression of some genes increased through TB treatment, whereas others decreased: some were persistently more highly expressed in TB-DM and others in TB-only patients. Genes involved in innate immune responses, anti-microbial immunity and inflammation were significantly upregulated in people with TB-DM throughout treatment. The overall pattern of change was similar across clinical groups irrespective of diabetes status, permitting models predictive of TB treatment to be developed. Exacerbated transcriptome changes in TB-DM take longer to resolve during TB treatment, meaning they remain different from those in uncomplicated TB after treatment completion. This may indicate a prolonged inflammatory response in TB-DM, requiring prolonged treatment or host-directed therapy for complete cure. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes for use across populations.

Neutrophil extracellular traps: from antimicrobial innate immunity to the development of chemotherapy-induced peripheral neuropathy.

Neutrophil extracellular traps: from antimicrobial innate immunity to the development of chemotherapy-induced peripheral neuropathy.

P069 Elevated adaptive immune responses to multiple Lachnospiraceae bacterial flagellins in therapy-naïve pediatric CD patients are associated with distinct immune pathology.

Abstract Background In inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), CD4+ T cell-responses to unknown microbial antigens drive intestinal inflammation. Both hypo- or hyperactive anti-microbial innate immunity may underlie these pathogenic T-cell responses. Previously, IgG and T-cell responses to Lachnospiraceae flagellins were detected in spontaneously colitic mice with an innate immune defect and adult CD patients with complicated disease. However, whether these IgG responses occur in therapy-naïve pediatric patients and whether they relate to hypo- or hyperresponsiveness is unknown. We hypothesize that, at diagnosis, a subgroup of pediatric CD patients has elevated anti-Lachnospiraceae flagellin IgG and concomitant inflammatory flagellin-specific T-cell responses associating with insufficient anti-microbial innate immunity. Methods Plasma IgG reactivity to 20 flagellins from Lachnospiraceae that colonize mouse and human gut was measured in therapy-naïve pediatric IBD patients (CD n=49; UC n=16), non-IBD (n=12) and age-matched healthy controls (HC n=17). CD patients were divided in two groups: ‘flagellin-hi’ (elevated IgG to 11-20 flagellins) and ‘flagellin-lo’ (elevated IgG to 0-10 flagellins), whose immunological and clinical profiles were compared. Results The proportion of flagellin-hi patients was higher in CD (41%) compared to UC (6%), non-IBD (8%) and HC (6%). In CD patients, clinical disease activity score, CRP and ESR did not relate to flagellin reactivity. Compared to flagellin-lo patients, flagellin-hi patients had increased frequencies of inflammatory gut-homing (CD38+CD62Lneg) circulating CD4+ T cells. Crucially, flagellin-hi patients had high frequencies of circulating flagellin-specific CD4+ T cells. Although high anti-flagellin IgG reactivity associated with increased flagellin-specific- and gut-homing- T-cell frequencies in the circulation, it associated with very limited immune cell infiltration, epithelial damage and phosphorylated NFκB in colonic biopsies on histology. Moreover, the plasma concentration of EN-RAGE, a neutrophil-derived protein, was decreased in flagellin-hi compared -lo patients. Conclusion In sum, a subgroup of pediatric therapy-naïve CD patients has high IgG responses to Lachnospiraceae-derived flagellins. While these flagellin-hi patients have higher frequencies of circulating gut-homing and flagellin-specific CD4+ T cells, they have lower colonic histological disease activity and NFκB activation, possibly suggesting insufficient innate immune cell activation and/or infiltration. Together, our data raise the question whether the enhanced anti-Lachnospiraceae adaptive immune response in a subgroup of CD patients may be driven by insufficient local innate immunity.

ETIOLOGICAL FACTORS IN DISEASES OF HARD TISSUES AND PERIODONTIUM IN PREGNANT WOMEN

Pregnant women have one of the highest risks of dental disease [3]. According to a number of authors, during the physiological course of pregnancy, the prevalence of dental caries is 91.4%, periodontal tissue diseases occur in 90% of cases, lesions of previously intact teeth with a predominantly acute course of the carious process - in 38% of pregnant patients [5 ]. Secondary caries, progression of the carious process, enamel hyperesthesia occurs in 79% of pregnant women [11]. At the same time, the intensity of the increase in dental caries in terms of the absolute increase in the index of caries-filling-removal during the gestational period is 0.83 [2]. In 50% of pregnant women and women in labor, the so-called gingivitis of pregnant women is observed during the normal course of the gestational period already at 2-3 months of pregnancy [12]. From the second half of pregnancy, the pathological process becomes more pronounced and more often proceeds as a generalized catarrhal or hypertrophic gingivitis, and pyogenic granuloma often develops. As pregnancy progresses, periodontal disease progresses continuously, and only in the postpartum period does the clinical picture improve [4]. In the long term, gingivitis that occurs during pregnancy becomes chronic [9, 10]. In a pregnant woman, against the background of altered reactivity and reduced body resistance, latent odontogenic foci of infection can lead to serious complications as a result of exacerbation of the inflammatory process [7, 8]. The greatest severity of inflammatory phenomena in periodontal tissues occurs in the second trimester of pregnancy, and the critical increase in the cariogenic situation in the oral cavity occurs in the third trimester [4, 2], which not only determines the optimal timing of dental examinations during pregnancy and the postpartum period, but also the differentiation of the approach to programs for the prevention and treatment of the most significant diseases of the oral cavity for this period of pregnancy. The decisive role in the development of caries and periodontitis in pregnant women belongs to hormonal substances (somatomammotropin, progesterone, gonadotropin) produced by the placenta, changes in mineral and protein metabolism, immunological status, oral microflora [1, 5]. The timing of the increased risk of the onset and progression of periodontal diseases, as well as the increase in the intensity of dental caries in pregnant women, is not enough to state; tires. This does not negate the significance of already established factors affecting the occurrence and development of dental caries, periodontal disease during pregnancy, but is aimed at competently combining diagnostically important known and newly identified criteria into a system of practical recommendations for identifying risk groups among pregnant women. on dental health and special monitoring of them. Immunoprotective peptides of biological media, including oral fluid, are markers of the intensity of local inflammation and are responsible for the implementation of innate antimicrobial immunity [6, 12]. In the oral fluid, a whole complex of immunoprotective peptides is isolated, among which lactoferrin, cathelicidin LL-37, and α-defensin are distinguished [14]. The inclusion of immunoprotective peptides in algorithms and models for ranking the risk of progression of dental caries during pregnancy will expand the boundaries of existing recommendations in this direction. When studying systemic immunity in pregnant women with periodontal diseases, oral fluid and peripheral blood are used as biological media [8]. At the same time, retroplacental blood and umbilical cord blood containing fetal and maternal blood, despite the easy method of selection after the birth of a child and placenta, not associated with invasive manipulations, is not used as a biological medium. Meanwhile, the determination of the spectrum of inflammatory mediators, the mineral composition of retroplacental and umbilical cord blood will allow answering the questions whether inflammation of the periodontal tissues of varying severity was accompanied by the "mother-placenta-fetus" system, whether it is possible to predict from the stage of childbirth in the future the appearance of caries of milk teeth? The purpose of this study is to optimize the system of providing dental care to pregnant women using clinical and laboratory indicators.

Optimization of growth of Levilactobacillus brevis SP 48 and in vitro evaluation of the effect of viable cells and high molecular weight potential postbiotics on Helicobacter pylori.

Several Levilactobacillus brevis strains have the potential to be used as probiotics since they provide health benefits due to the interaction of live cells, and of their secreted products, with the host (tissues). Therefore, the development of simple fermentation processes that improve cell viability to reduce industrial production costs, and at the same time the characterization and biological evaluation of cell-free postbiotics that can further promote application, are of great interest. In the present study, small scale batch fermentations on semi defined media, deprived of animal derived raw materials, were used to optimize growth of L. brevis SP48, reaching 1.2 ± 0.4 × 1010CFU/ml of viable cells after 16h of growth. Displacement, competition, and inhibition assays compared the effect, on Helicobacter pylori, of L. brevis cells to that of its partially purified potentially postbiotic fraction rich in exopolysaccharides and proteins. The expression of pro and anti-inflammatory biochemical markers indicated that both samples activated antimicrobial defenses and innate immunity in a gastric model. Moreover, these compounds also acted as modulators of the inflammatory response in a gut in vitro model. These data demonstrate that the high molecular weight compounds secreted by L. brevis SP48 can contrast H. pylori and reduce inflammation related to intestinal bowel disease, potentially overcoming issues related to the preservation of probiotic viability.

Association of peptidoglycan recognition protein 1 to post-myocardial infarction and periodontal inflammation: A subgroup report from the PAROKRANK (Periodontal Disease and the Relation to Myocardial Infarction) study.

Peptidoglycan recognition protein 1 (PGLYRP1) is an antimicrobial and proinflammatory innate immunity protein activated during infections. We aimed to investigate whether PGYLRP1 and associated molecules of the immune response in saliva is a cumulative outcome result of both MI and periodontal inflammation. Two hundred patients with MI and another 200 matched non-MI controls were included. A full-mouthexamination was conducted to assess periodontal inflammation and collection of stimulated saliva was performed 6 to 10weeks after the first MI. PGLYRP1, triggering receptor expressed on myeloid cells 1(TREM-1), interleukin-1 beta (IL-1β) were analyzed by ELISA. Matrix metalloproteinase (MMP)-8 levels were determined by IFMA. Compared to controls, MI patients showed higher salivary PGLYRP1, but not TREM-1 levels. The difference in PGLYRP1 levels remained after adjustment for covariates. In MI patients, the PGLYRP1 levels positively correlated with BOP and PPD 4 to 5mm. Among non-MI subjects, the levels of PGLYRP1 correlated positively and significantly with BOP and total PPD. Salivary PGLYRP1 concentrations also showed strong positive correlations with levels of TREM-1, IL-1β and MMP-8. In multivariate linear regression analysis, in MI patients, BOP and former smokingstatus displayed an association with salivary PGLYRP1 concentration. MI patients showed higher salivary PGLYRP1 levels than healthy controls, also after adjusting for smoking, sex, age and periodontal health status. Salivary levels of PGLYRP1 may reflect the overall inflammatory burden to chronic bacterial exposure, possibly underpinning the observed associations between periodontitis and exposure with MI.

Role of Vγ9vδ2 T lymphocytes in infectious diseases.

The T cell receptor Vγ9Vδ2 T cells bridge innate and adaptive antimicrobial immunity in primates. These Vγ9Vδ2 T cells respond to phosphoantigens (pAgs) present in microbial or eukaryotic cells in a butyrophilin 3A1 (BTN3) and butyrophilin 2A1 (BTN2A1) dependent manner. In humans, the rapid expansion of circulating Vγ9Vδ2 T lymphocytes during several infections as well as their localization at the site of active disease demonstrates their important role in the immune response to infection. However, Vγ9Vδ2 T cell deficiencies have been observed in some infectious diseases such as active tuberculosis and chronic viral infections. In this review, we are providing an overview of the mechanisms of Vγ9Vδ2 T cell-mediated antimicrobial immunity. These cells kill infected cells mainly by releasing lytic mediators and pro-inflammatory cytokines and inducing target cell apoptosis. In addition, the release of chemokines and cytokines allows the recruitment and activation of immune cells, promoting the initiation of the adaptive immune response. Finaly, we also describe potential new therapeutic tools of Vγ9Vδ2 T cell-based immunotherapy that could be applied to emerging infections.

Skin Injury Activates a Rapid TRPV1-Dependent Antiviral Protein Response

The skin serves as the interface between the body and the environment and plays a fundamental role in innate antimicrobial host immunity. Antiviral proteins (AVPs) are part of the innate host defense system and provide protection against viral pathogens. How breach of the skin barrier influences innate AVP production remains largely unknown. In this study, we characterized the induction and regulation of AVPs after skin injury and identified a key role of TRPV1 in this process. Transcriptional and phenotypic profiling of cutaneous wounds revealed that skin injury induces high levels of AVPs in both mice and humans. Remarkably, pharmacologic and genetic ablation of TRPV1-mediated nociception abrogated the induction of AVPs, including Oas2, Oasl2, and Isg15 after skin injury in mice. Conversely, stimulation of TRPV1 nociceptors was sufficient to induce AVP production involving the CD301b+ cells‒IL-27‒mediated signaling pathway. Using IL-27 receptor‒knockout mice, we show that IL-27 signaling is required in the induction of AVPs after skin injury. Finally, loss of TRPV1 signaling leads to increased viral infectivity of herpes simplex virus. Together, our data indicate that TRPV1 signaling ensures skin antiviral competence on wounding.

Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

Multiplexed proteomics of autophagy deficient macrophages reveals enhanced anti-microbial immunity via the oxidative stress response

Abstract Macrophages play a critical role in clearance of cytosolic pathogens. Autophagy functions at the intersection of antimicrobial innate immunity, metabolism and protein quality control; however, the impacts of infection and autophagy on shaping the macrophage proteome are poorly understood. Here, we describe a deep multi-dimensional proteomic analysis of primary murine macrophages infected with Shigella flexneri (S. flexneri). Tandem mass tagging (TMT) revealed dynamic genotype- and infection-dependent differences in host and pathogen proteins, phosphorylation and ubiquitination. These data catalogue the complex circuitry connecting autophagy, inflammatory signaling and the oxidative stress response. Loss of the autophagy gene Atg16l1 induced basal oxidative stress, activated the compensatory glutathione biosynthetic machinery, and surprisingly, enhanced clearance of S. flexneri. Pathogen clearance was similarly enhanced in wild type macrophages upon pharmacological inhibition of cysteine import. Our study provides a resource for innate immunity research and unexpectedly reveals that ATG16L1 dampens antimicrobial immunity by regulating oxidative stress.

Perillaldehyde Inhibition of cGAS Reduces dsDNA-Induced Interferon Response.

Cyclic GMP-AMP synthase (cGAS), serving as a primary sensor of intracellular DNA, is essential to initiate anti-microbial innate immunity. Inappropriate activation of cGAS by self-DNA promotes severe autoinflammatory diseases such as Aicardi–Goutières syndrome (AGS); thus, inhibition of cGAS may provide therapeutic benefit in anti-autoimmunity. Here we report that perillaldehyde (PAH), a natural monoterpenoid compound derived from Perilla frutescens, suppresses cytosolic-DNA-induced innate immune responses by inhibiting cGAS activity. Mice treated with PAH are more susceptible to herpes simplex virus type 1 (HSV-1) infection. Moreover, administration with PAH markedly ameliorates self-DNA-induced autoinflammatory responses in a mouse model of AGS. Collectively, our study reveals that PAH can effectively inhibit cGAS-STING signaling and could be developed toward the treatment of cGAS-mediated autoimmune diseases.

The Butyrate-Producing Bacterium Clostridium butyricum Suppresses Clostridioides difficile Infection via Neutrophil- and Antimicrobial Cytokine-Dependent but GPR43/109a-Independent Mechanisms.

Short-chain fatty acids, such as butyrate, are major gut microbial metabolites that are beneficial for gastrointestinal health. Clostridium butyricum MIYAIRI588 (CBM588) is a bacterium that produces a robust amount of butyrate and therefore has been used as a live biotherapeutic probiotic in clinical settings. Clostridioides difficile causes life-threatening diarrhea and colitis. The gut resident microbiota plays a critical role in the prevention of C. difficile infection (CDI), as the disruption of the healthy microbiota by antibiotics greatly increases the risk for CDI. We report that CBM588 treatment in mice significantly improved clinical symptoms associated with CDI and increased the number of neutrophils and Th1 and Th17 cells in the colonic lamina propria in the early phase of CDI. The protective effect of CBM588 was abolished when neutrophils, IFN-γ, or IL-17A were depleted, suggesting that induction of the immune reactants is required to elicit the protective effect of the probiotic. The administration of tributyrin, which elevates the concentration of butyrate in the colon, also increased the number of neutrophils in the colonic lamina propria, indicating that butyrate is a potent booster of neutrophil activity during infection. However, GPR43 and GPR109a, two G protein-coupled receptors activated by butyrate, were dispensable for the protective effect of CBM588. These results indicate that CBM588 and butyrate suppress CDI, in part by boosting antimicrobial innate and cytokine-mediated immunity.

Abstract PO-089: Profiling of salivary proteome specific for the diagnosis of head and neck cancer among African Americans

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is one the types of Cancer in The United States with inequality in the mortality rate among Black patients when compared to White patients. Patients of European dissent (Whites) are more likely to be diagnosed with any type of HNSCC but patients of African dissents (Blacks) are more likely to die from ant type of HNSCC when all patients are HPV negative. The difference in survival based on race and ethnicity prompted the need for a better and early diagnostic marker for HNSCC among Blacks and other groups with poor prognosis of HNSCC. The present research seeks to identify a specific and safe salivary proteome marker for the early diagnosis of HNSCC. The hypothesis of this article is that there is a significant difference in the salivary proteome of AA patients with HNSCC when compared to WA patients which is specific to cancer site. Eight patient samples with confirmed diagnosis of Laryngeal cancer at Nashville General hospital (IRB Protocol Number 14-03-172) were collected with appropriate informed consent. Patient samples were pooled together and analyzed using IHC for the presence of shared protein expression. Each protein sample was later analyzed individually using IHC in order to obtain specific proteins expressed by each patient’s salivary proteome. A Multidimensional Protein Identification Technology (MUDPIT) was used to analyze salivary samples to increase resolution for identifying proteins and peptides. MUDPIT was used to increase resolution of analyzed proteins and was compared with other proteins associated with LaCa using a known online protein database called WebGestalt. The most highly expressed proteins in each of the samples were further analyzed using The Human Protein Atlas in order to study the location of the salivary proteome and its function when expressed. 20 unique protein genes obtained from Label Free Quantification (LFQ) analysis was further analyzed for the function of the genes and gene co-localization in the body using an online database known as GeneMANIA. Finally, PEAKS was used to ascertain the effect of proteins. Result shows differences in the salivary proteome of AA patients compared to WA’s. Specifically, proteins involved in transcriptional mis-regulation were over- represented in AA>WA patients which included some proteins associated with metastasis (JUP), dis-functional immune cells (CD14), over-expressed tumor- suppressor proteins (DMBT1 and DRB2) and elevated level of antimicrobial innate immunity (His 1 & 3) are all higher than the normal values in healthy patients without LaCa both among AA and WA. Future research is required to expand on these identified proteins to help provide a safe and specific biomarker for the diagnosis of LaCa among AA patients. Future research can also provide direction as to where therapeutics can be directed and how they can be safely administered to generate the most effective treatment that can improve the prognosis of the disease among AA patients and others with groups with poor prognosis. Citation Format: Oyinloye A. Jose, Oladipupo Anibire, Derek Wagner, Dana Marshall, Billy Ballard, Siddharth Pratap, Victor Paramov. Profiling of salivary proteome specific for the diagnosis of head and neck cancer among African Americans [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-089.

Bacterial but not fungal challenge up‐regulates the transcription of Coleoptericin genes in Tenebrio molitor

AbstractAntimicrobial peptides (AMPs) are effector candidates that elicit humoral immunity in invertebrates. AMPs facilitate bacterial clearance by either physically disrupting the microbial membranes or the intracellular targets. In the Coleopteran pest, Tenebrio molitor, transcriptional regulation of AMPs has been studied in the context of innate immune signaling cascades and antimicrobial immunity. Here, we report the transcriptional response of three AMP genes, Coleoptericin A, B, and C (TmCole A, B and C) in T. molitor in response to bacterial (Escherichia coli, Staphylococcus aureus), and fungal (Candida albicans) challenges. These genes were expressed essentially in the gut and hemocytes followed by the integument tissue of the T. molitor larva. Further, these genes were highly expressed in the late‐larval, pupal, and early adult stages. Furthermore, while all of these transcripts were highly upregulated in the fat body and Malpighian tubules after bacterial challenge, TmCole B and TmCole C were induced in the gut after E. coli challenge. Fungal stimulation was not required for the upregulation of the transcription of Coleoptericin genes in T. molitor.

The Hippo Pathway in Innate Anti-microbial Immunity and Anti-tumor Immunity.

The Hippo pathway responds to diverse environmental cues and plays key roles in cell fate determination, tissue homeostasis, and organ regeneration. Aberrant Hippo signaling, on the other hand, has frequently been implicated in diversified pathologies such as cancer and immune dysfunction. Here, we summarize the recent but rapid progress in understanding the involvement of the Hippo pathway in innate immunity, with a special focus on the intrinsic mechanisms and mutual interactions between Hippo-YAP signaling and the innate immune response and its physiological impacts on anti-microbial immunity and anti-tumor immunity. Moving forward, we believe that systematic investigations under the physiological setting are needed to draw a clearer picture of the actions of Hippo in innate immunity.

Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease.

Background: Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen and have a highly specialised but plastic phenotype that is governed by signals in the local microenvironment. AMs are thought to maintain immunological homeostasis in the steady state, but have also been implicated in the pathogenesis of allergic airway disease (AAD). In this study, we aimed to better understand these potentially contrasting AM functions using transcriptomic analysis. Methods: Bulk RNA sequencing was performed on AMs (CD11c + Siglec F + CD64 + CD45 + SSC hi) flow cytometry sorted from C57BL/6 mice during experimental AAD driven by repeated house dust mite inhalation (AMs HDM), compared to control AMs from non-allergic mice. Differentially expressed genes were further analysed by hierarchical clustering and biological pathway analysis. Results: AMs HDM showed increased expression of genes associated with antigen presentation, inflammatory cell recruitment and tissue repair, including several chemokine and matrix metalloproteinase genes. This was accompanied by increased expression of mitochondrial electron transport chain subunit genes and the retinoic acid biosynthetic enzyme gene Raldh2. Conversely, AMs HDM displayed decreased expression of a number of cell cycle genes, genes related to cytoskeletal functions and a subset of genes implicated in antimicrobial innate immunity, such as Tlr5, Il18 and Tnf. Differential gene expression in AMs HDM was consistent with upstream effects of the cytokines IL-4 and IFN-γ, both of which were present at increased concentrations in lung tissue after HDM treatment. Conclusions: These data highlight diverse gene expression changes in the total AM population in a clinically relevant mouse model of AAD, collectively suggestive of contributions to inflammation and tissue repair/remodelling, but with decreases in certain steady state cellular and immunological functions.