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Riemerella anatipestifer (RA) is a significant poultry pathogen causing acute septicemia and inflammation. The function of protease RAYM_01812, responsible for gelatin degradation, is unexplored in RA pathogenesis. To elucidate its role, we generated a deletion mutant ΔRAYM_01812 (ΔRAYM) and complementary CΔRAYM_01812 (CΔRAYM) strain and revealed the protease's role in extracellular gelatinase activity. By expressing full-length 76 kDa RAYM_01812 protein without signal peptide as well as seven partial structural domains fragments, we evidence that the N-terminal propeptide acts as an enzymatic activity inhibitor and it gets cleaved at A 112 . Also, we show that the β-fold sheet domain is necessary for enhancing the enzymatic protease activity. Sequential auto-proteolysis forms a stable 40 kDa enzyme. Then, testing the strains in duck sera indicated that the absence or presence of RAYM_01812 results in reduced or enhanced bacterial survival, respectively. Furthermore, we found that the protease is able to cleave IgY antibodies as well as the complement factors C3a and C5a, that the protease reduces C3a- or C5a-mediated monocyte chemotaxis, and results in enhanced membrane attack complex (MAC) formation on the surface of ΔRAYM compared to CΔRAYM. This suggests that RAYM_01812 plays a crucial role in protecting against the serum complement-mediated bactericidal effect through inhibiting MAC formation and monocyte chemotaxis. Animal infection assays showed a 1090-fold reduced virulence of ΔRAYM compared to RA-YM, evidenced by decreased tissue loading and weaker histopathological changes. In conclusion, RAYM_01812 acts as a vital virulence factor, enabling host innate immune defence escape through complement killing evasion and monocyte chemotaxis inhibition.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic β-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2'O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8., (© 2024. The Author(s).)

The accidental human pathogen Legionella pneumophila (Lp) is the etiological agent for a severe atypical pneumonia known as Legionnaires' disease. In human infections and animal models of disease alveolar macrophages are the primary cellular niche that supports bacterial replication within a unique intracellular membrane-bound organelle. The Dot/Icm apparatus-a type IV secretion system that translocates ~300 bacterial proteins within the cytosol of the infected cell-is a central virulence factor required for intracellular growth. Mutant strains lacking functional Dot/Icm apparatus are transported to and degraded within the lysosomes of infected macrophages. The early foundational work from Dr. Horwitz's group unequivocally established that Legionella does not replicate extracellularly during infection-a phenomenon well supported by experimental evidence for four decades. Our data challenges this paradigm by demonstrating that macrophages and monocytes provide the necessary nutrients and support robust Legionella extracellular replication. We show that the previously reported lack of Lp extracellular replication is not a bacteria intrinsic feature but rather a result of robust restriction by serum-derived nutritional immunity factors. Specifically, the host iron-sequestering protein Transferrin is identified here as a critical suppressor of Lp extracellular replication in an iron-dependent manner. In iron-overload conditions or in the absence of Transferrin, Lp bypasses growth restriction by IFNγ-primed macrophages though extracellular replication. It is well established that certain risk factors associated with development of Legionnaires' disease, such as smoking, produce a chronic pulmonary environment of iron-overload. Our work indicates that iron-overload could be an important determinant of severe infection by allowing Lp to overcome nutritional immunity and replicate extracellularly, which in turn would circumvent intracellular cell intrinsic host defenses. Thus, we provide evidence for nutritional immunity as a key underappreciated host defense mechanism in Legionella pathogenesis., (© 2024. The Author(s).)

Bordetella pertussis infects the upper airways of humans and disarms host defense by the potent immuno-subversive activities of its pertussis (PT) and adenylate cyclase (CyaA) toxins. CyaA action near-instantly ablates the bactericidal activities of sentinel CR3-expressing myeloid phagocytes by hijacking cellular signaling pathways through the unregulated production of cAMP. Moreover, CyaA-elicited cAMP signaling also inhibits the macrophage colony-stimulating factor (M-CSF)-induced differentiation of incoming inflammatory monocytes into bactericidal macrophages. We show that CyaA/cAMP signaling via protein kinase A (PKA) downregulates the M-CSF-elicited expression of monocyte receptors for transferrin (CD71) and hemoglobin-haptoglobin (CD163), as well as the expression of heme oxygenase-1 (HO-1) involved in iron liberation from internalized heme. The impact of CyaA action on CD71 and CD163 levels in differentiating monocytes is largely alleviated by the histone deacetylase inhibitor trichostatin A (TSA), indicating that CyaA/cAMP signaling triggers epigenetic silencing of genes for micronutrient acquisition receptors. These results suggest a new mechanism by which B. pertussis evades host sentinel phagocytes to achieve proliferation on airway mucosa.IMPORTANCETo establish a productive infection of the nasopharyngeal mucosa and proliferate to sufficiently high numbers that trigger rhinitis and aerosol-mediated transmission, the pertussis agent Bordetella pertussis deploys several immunosuppressive protein toxins that compromise the sentinel functions of mucosa patrolling phagocytes. We show that cAMP signaling elicited by very low concentrations (22 pM) of Bordetella adenylate cyclase toxin downregulates the iron acquisition systems of CD14 + monocytes. The resulting iron deprivation of iron, a key micronutrient, then represents an additional aspect of CyaA toxin action involved in the inhibition of differentiation of monocytes into the enlarged bactericidal macrophage cells. This corroborates the newly discovered paradigm of host defense evasion mechanisms employed by bacterial pathogens, where manipulation of cellular cAMP levels blocks monocyte to macrophage transition and replenishment of exhausted phagocytes, thereby contributing to the formation of a safe niche for pathogen proliferation and dissemination., Competing Interests: The authors declare no conflict of interest.

Candida albicans, an opportunistic fungal pathogen, produces the quorum-sensing molecule farnesol, which we have shown alters the transcriptional response and phenotype of human monocyte-derived dendritic cells (DCs), including their cytokine secretion and ability to prime T cells. This is partially dependent on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which has numerous ligands, including the sphingolipid metabolite sphingosine 1-phosphate. Sphingolipids are a vital component of membranes that affect membrane protein arrangement and phagocytosis of C. albicans by DCs. Thus, we quantified sphingolipid metabolites in monocytes differentiating into DCs by High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Farnesol increased the activity of serine palmitoyltransferase, leading to increased levels of 3-keto-dihydrosphingosine, dihydrosphingosine, and dihydrosphingosine 1-phosphate and inhibited dihydroceramide desaturase by inducing oxidative stress, leading to increased levels of dihydroceramide and dihydrosphingomyelin species and reduced ceramide levels. Accumulation of dihydroceramides can inhibit mitochondrial function; accordingly, farnesol reduced mitochondrial respiration. Dihydroceramide desaturase inhibition increases lipid droplet formation, which we observed in farnesol-treated cells, coupled with an increase in intracellular triacylglycerol species. Furthermore, inhibition of dihydroceramide desaturase with either farnesol or specific inhibitors impaired the ability of DCs to prime interferon-γ-producing T cells. The effect of farnesol on sphingolipid metabolism, triacylglycerol synthesis, and mitochondrial respiration was not dependent on PPAR-γ. In summary, our data reveal novel effects of farnesol on sphingolipid metabolism, neutral lipid synthesis, and mitochondrial function in DCs that affect their instruction of T cell cytokine secretion, indicating that C. albicans can manipulate host cell metabolism via farnesol secretion.IMPORTANCE Candida albicans is a common commensal yeast, but it is also an opportunistic pathogen which is one of the leading causes of potentially lethal hospital-acquired infections. There is growing evidence that its overgrowth in the gut can influence diseases as diverse as alcohol-associated liver disease and COVID-19. Previously, we found that its quorum-sensing molecule, farnesol, alters the phenotype of dendritic cells differentiating from monocytes, impairing their ability to drive protective T cell responses. Here, we demonstrate that farnesol alters the metabolism of sphingolipids, important structural components of the membrane that also act as signaling molecules. In monocytes differentiating to dendritic cells, farnesol inhibited dihydroceramide desaturase, resulting in the accumulation of dihydroceramides and a reduction in ceramide levels. Farnesol impaired mitochondrial respiration, known to occur with an accumulation of dihydroceramides, and induced the accumulation of triacylglycerol and oil bodies. Inhibition of dihydroceramide desaturase resulted in the impaired ability of DCs to induce interferon-γ production by T cells. Thus, farnesol production by C. albicans could manipulate the function of dendritic cells by altering the sphingolipidome., Competing Interests: The authors declare no conflict of interest.

Objective: The health of oral cavity is considered as an important indicator of aging. Oral microbiota is highly associated with the oral health, while the variation of oral microbiome in elderly population and characteristic microbes associated with aging remain unclear., Subjects and Methods: In this study, 130 elderly subjects were recruited and divided into 3 groups according to their age: Stage I group (65 ≤ years < 70), Stage II group (70 ≤ years < 75), and Stage III group (75 ≤ years < 80). Their physiological indices were analyzed with using Illumina MiSeq platform and the oral microbiome was determined by high-throughput sequencing., Results: Along with aging, the level of fasting blood glucose, systolic pressure and monocytes are significantly increased. No significant difference was detected on the whole structure of the oral microbiome among groups. While using Metastats and Spearman's correlation analysis, specific bacteria were identified as potential age- or health index-related bacterial genera including Fusobacterium, Parvimonas, Porphyromonas, Aminobacter, Collinsella, Clostridium and Acinetobacter., Conclusion: Our study revealed that the composition structure of salivary microbiota in elderly population was relatively stable while specific bacteria were correlated with age and health status, which is promising to be served as health indicators of the elderly after further exploration., (© 2024. The Author(s).)

Introduction: Streptococcus pneumoniae (the pneumococcus) effectively colonizes the human nasopharynx, but can migrate to other host sites, causing infections such as pneumonia and sepsis. Previous studies indicate that pneumococci grown as biofilms have phenotypes of bacteria associated with colonization whereas bacteria released from biofilms in response to changes in the local environment (i.e., dispersed bacteria) represent populations with phenotypes associated with disease. How these niche-adapted populations interact with immune cells upon reaching the vascular compartment has not previously been studied. Here, we investigated neutrophil, monocyte, and platelet activation using ex vivo stimulation of whole blood and platelet-rich plasma with pneumococcal populations representing distinct stages of the infectious process (biofilm bacteria and dispersed bacteria) as well as conventional broth-grown culture (planktonic bacteria)., Methods: Flow cytometry and ELISA were used to assess surface and soluble activation markers for neutrophil and monocyte activation, platelet-neutrophil complex and platelet-monocyte complex formation, and platelet activation and responsiveness., Results: Overall, we found that biofilm-derived bacteria (biofilm bacteria and dispersed bacteria) induced significant activation of neutrophils, monocytes, and platelets. In contrast, little to no activation was induced by planktonic bacteria. Platelets remained functional after stimulation with bacterial populations and the degree of responsiveness was inversely related to initial activation. Bacterial association with immune cells followed a similar pattern as activation., Discussion: Differences in activation of and association with immune cells by biofilm-derived populations could be an important consideration for other pathogens that have a biofilm state. Gaining insight into how these bacterial populations interact with the host immune response may reveal immunomodulatory targets to interfere with disease development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chao, Mørch, Håkansson and Shannon.)

Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1β and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Khongpraphan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis -containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech&#95;1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech&#95;1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients., Competing Interests: The authors have declared that no competing interests exist.

Shigellosis causes most diarrheal deaths worldwide, particularly affecting children. Shigella invades and replicates in the epithelium of the large intestine, eliciting inflammation and tissue destruction. To understand how Shigella rewires macrophages prior to epithelium invasion, we performed genome-wide and focused secondary CRISPR knockout and CRISPR interference (CRISPRi) screens in Shigella flexneri-infected human monocytic THP-1 cells. Knockdown of the Toll-like receptor 1/2 signaling pathway significantly reduced proinflammatory cytokine and chemokine production, enhanced host cell survival, and controlled intracellular pathogen growth. Knockdown of the enzymatic component of the mitochondrial pyruvate dehydrogenase complex enhanced THP-1 cell survival. Small-molecule inhibitors blocking key components of these pathways had similar effects; these were validated with human monocyte-derived macrophages, which closely mimic the in vivo physiological state of macrophages postinfection. High-throughput CRISPR screens can elucidate how S. flexneri triggers inflammation and redirects host pyruvate catabolism for energy acquisition before killing macrophages, pointing to new shigellosis therapies. IMPORTANCE Treatment for shigellosis is becoming increasingly difficult as resistance to antibiotics becomes more prevalent. One way to prevent this significant public health problem from developing into a full-blown crisis is to approach shigellosis intervention from the point of view of the host. So far, little is known about the specific biological pathways that might be modulated in macrophages, sentinel cells of the innate immune system, to strengthen the response to Shigella infection. In this work, we conducted CRISPR screens to comprehensively decipher the complexity of macrophage- Shigella interactions and to discover new potential therapeutic interventions against Shigella flexneri infection. Our work highlights systematic genetic perturbation strategies to provide direct causal evidence showing how intracellular pathogens manipulate innate immune cells.

Introduction: Q fever, a zoonosis caused by Coxiella burnetii , affects more males than females despite a similar level of exposure. A protective role of estradiol has been reported in mice, suggesting that sex hormones are involved in C. burnetii infection. We wondered whether the responses of monocytes and monocyte-derived macrophages (MDMs) to C. burnetii are influenced by sex hormones., Materials and Methods: The bacterial intracellular fate in monocytes was studied using quantitative PCR, and monocyte cytokine production in response to C. burnetii was assessed using qRT-PCR and immunoassays. Before infection, MDMs from males and females were incubated with testosterone and estradiol, respectively., Results: Bacterial uptake and persistence were similar in monocytes from males and females but were slightly increased in male MDMs. The expression of inflammatory genes, including those encoding TNF and CXCL10, was higher in MDMs from females than in MDMs from males infected by C. burnetii . Adding testosterone to male MDMs amplified their immunoregulatory properties, including increased expression of IL10 and TGFB genes and TGF-β production in response to C. burnetii. In contrast, adding estradiol to MDMs from females had no effect on their inflammatory profile., Conclusion: The stronger inflammatory profile of macrophages from females may have a protective role, likely under estrogen control, while testosterone may affect disease progression by promoting an anti-inflammatory response. This finding may have consequences for personalized management of patients with Q fever., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gay, Melenotte, Lopez, Desnues, Raoult, Leone, Mezouar and Mege.)

Immune checkpoints play various important roles in tumour immunity, which usually contribute to T cells' exhaustion, leading to immunosuppression in the tumour microenvironment. However, the roles of immune checkpoints in infectious diseases, especially fungal infection, remain elusive. Here, we reanalyzed a recent published single-cell RNA-sequencing (scRNA-seq) data of peripheral blood mononuclear cells (PBMCs) stimulated with Candida albicans ( C. albicans ), to explore the expression patterns of immune checkpoints after C. albicans bloodstream infection. We characterized the heterogeneous pathway activities among different immune cell subpopulations after C. albicans infection. The CTLA-4 pathway was up-regulated in stimulated CD4 + and CD8 + T cells, while the PD-1 pathway showed high activity in stimulated plasmacytoid dendritic cell (pDC) and monocytes. Importantly, we found that immunosuppressive checkpoints HAVCR2 and LAG3 were only expressed in stimulated NK and CD8 + T cells, respectively. Their viabilities were validated by flow cytometry. We also identified three overexpressed genes ( ISG20 , LY6E , ISG15 ) across all stimulated cells. Also, two monocyte-specific overexpressed genes ( SNX10 , IDO1 ) were screened out in this study. Together, these results supplemented the landscape of immune checkpoints in fungal infection, which may serve as potential therapeutic targets for C. albicans infection. Moreover, the genes with the most relevant for C. albicans infection were identified in this study.

Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Morante-Palacios, Lorente-Sorolla, Ciudad, Calafell-Segura, Garcia-Gomez, Català-Moll, Ruiz-Sanmartín, Martínez-Gallo, Ferrer, Ruiz-Rodriguez, Álvarez-Errico and Ballestar.)

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis ( Mtb ), and 5%-10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb -infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Horváti, Fodor, Pályi, Henczkó, Balka, Gyulai, Kiss, Biri-Kovács, Senoner and Bősze.)

Innate immune memory, the ability of innate cells to react in a more protective way to secondary challenges, is induced by exposure to infectious and other exogeous and endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as such, could trigger an inflammatory reaction in monocytes and macrophages and could therefore be also able to induce innate memory. Here, we have evaluated the capacity of engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the memory responses induced by microbial agents. Microbial agents used were in soluble vs . particulate form (MDP and the gram-positive bacteria Staphylococcus aureus ; β-glucan and the β-glucan-producing fungi C. albicans ), and as whole microrganisms that were either killed ( S. aureus , C. albicans ) or viable (the gram-negative bacteria Helicobacter pylori ). The memory response was assessed in vitro , by exposing human primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs (primary response), then resting them for 6 days to allow return to baseline, and eventually challenging them with LPS (secondary memory response). Primary and memory responses were tested as production of the innate/inflammatory cytokine TNFα and other inflammatory and anti-inflammatory factors. While inactive on the response induced by soluble microbial stimuli (muramyl dipeptide -MDP-, β-glucan), AuNPs partially reduced the primary response induced by whole microorganisms. AuNPs were also unable to directly induce a memory response but could modulate stimulus-induced memory in a circumscribed fashion, limited to some agents and some cytokines. Thus, the MDP-induced tolerance in terms of TNFα production was further exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory response. Conversely, the H. pylori -induced tolerance was downregulated by AuNPs only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more inflammatory memory response. These effects of AuNPs may depend on a differential interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Swartzwelter, Michelini, Frauenlob, Barbero, Verde, De Luca, Puntes, Duschl, Horejs-Hoeck, Italiani and Boraschi.)

Background: Sepsis is a critical medical condition that requires additional diagnostic considerations. Recently, focus has shifted to the diagnosis of sepsis using new markers to overcome the limitations of traditional laboratory diagnostic modalities. Neutrophil CD11b (nCD11b) and monocyteCD14 (mCD14) cell surface antigens have been shown to be useful in such diagnostic consideration., Aim: To investigate the diagnostic, monitoring, prognostic, and predictive roles of nCD11b and mCD14 as sepsis biomarkers in comparison to each other and to traditional laboratory sepsis parameters in order to select the best fit for routine daily use in neonatal intensive care units (NICUs)., Subject: The study included 188 neonates from Ain Shams University Hospitals' NICUs, who were divided into two groups: the control group ( n = 100) and the sepsis group ( n = 88). Highly sensitive CRP (hs-CRP), complete blood count (CBC), blood culture, and nCD11b and mCD14 evaluations were all part of the laboratory sepsis evaluation (done by flow cytometry technology). Positive blood culture results (BACT/ALERT system) confirmed the sepsis diagnosis. Twenty-four enrolled sepsis neonates were subjected to follow-up assessments, and they were divided into two groups based on clinical improvement: improved sepsis and sepsis without improvement. In order to predict performance evaluation, the subjected neonates were reclassified according to their outcome into survivors' and nonsurvivors' group., Results: Sepsis patients had a significant increase in mCD14 MFI values when compared to controls. With sensitivity 75.4 percent, specificity 71.9 percent, efficacy 73.3 percent, and AUC 0.703, mCD14 MFI at cutoff 9.36 could distinguish the presence of septicemia. Significant increases in both mCD14 MFI and nCD11b MFI ( P = 0.001) were observed in the severe sepsis/septic shock group compared to the nonsevere sepsis group. The combined measurement of CD14 MFI at cutoff 9.97 and CD14 percent at cutoff 44.7 percent yielded the best predictive performance., Conclusion: Sepsis patients had a significant increase in mCD14 MFI comparable to the controls. mCD14 MFI demonstrated better diagnostic, prognostic, and predictive results than nCD11b. hs-CRP outperformed mCD14 and nCD11b in terms of diagnostic efficacy and AUC. In the monitoring of sepsis patients, both mCD14 and nCD11b produced unsatisfactory results. Currently, the routine use of mCD14 or nCD11b as sepsis biomarkers in neonatal ICUs is not justified., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Heba E. Hashem et al.)

Aims: Periodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development., Materials and Methods: We combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2'-deoxy-2'-[ 18 F]fluoro-D-glucose positron-emission-tomography ([ 18 F]FDG PET/CT) scanning., Results: When adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [ 18 F]FDG PET/CT imaging showed a trend for increased [ 18 F]FDG-uptake in the periodontium [mean standard uptake value (SUV mean ), p=0.11] and in femur bone marrow (SUV mean , p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found., Conclusions: P. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation., Competing Interests: LJ and MGN are scientific founders of TTxD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Noz, Plachokova, Smeets, Aarntzen, Bekkering, Vart, Joosten, Netea and Riksen.)

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ 9 δ 2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ 9 δ 2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rasi, Wood, Eickhoff, Xia, Pozzi, Edwards, Walch, Bovenschen and Hoft.)

Most bacteria naturally release spherical lipid-bilayered extracellular vesicles (EVs) containing proteins, nucleic acids, and virulence-related molecules, thus contributing to diverse biological functions including transport of virulence factors. The group A streptococcus, Streptococcus pyogenes (GAS), a major human pathogen, also releases EVs; however, it remains unclear how GAS EVs interact physiologically and pathologically with host cells, and what the differences are between invasive and non-invasive strains. The proteome profile in this study revealed that GAS EVs enclosed many virulence-related proteins such as streptolysin O and NAD-glycohydrolase, facilitating their pathogenicity, and invasive GAS EVs were more abundant than non-invasive counterparts. In terms of biological effects, invasive GAS EVs showed slo -dependent cytotoxic activity and the induction of cytokine expression, contributing to GAS pathogenicity directly. Although non-invasive GAS EVs did not show cytotoxic activity, they may be utilized as a means to prevent antibacterial mechanisms such as autophagy, leading to enhancement of their own survival in the intracellular environment after the infection. These results suggest that invasive and non-invasive GAS EVs play different roles in GAS infection strategy and pathogenicity. Our findings also indicate that EVs could be a key factor for GAS pathogenicity in GAS-host interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Murase, Aikawa, Nozawa, Nakatake, Sakamoto, Kikuchi and Nakagawa.)

Orientia (O.) tsutsugamushi , the causative agent of scrub typhus, is a neglected, obligate intracellular bacterium that has a prominent tropism for monocytes and macrophages. Complications often involve the lung, where interstitial pneumonia is a typical finding. The severity of scrub typhus in humans has been linked to altered plasma concentrations of chemokines which are known to act as chemoattractants for myeloid cells. The trafficking and function of monocyte responses is critically regulated by interaction of the CC chemokine ligand 2 (CCL2) and its CC chemokine receptor CCR2. In a self-healing mouse model of intradermal infection with the human-pathogenic Karp strain of O. tsutsugamushi , we investigated the role of CCR2 on bacterial dissemination, development of symptoms, lung histology and monocyte subsets in blood and lungs. CCR2-deficient mice showed a delayed onset of disease and resolution of symptoms, higher concentrations and impaired clearance of bacteria in the lung and the liver, accompanied by a slow infiltration of interstitial macrophages into the lungs. In the blood, we found an induction of circulating monocytes that depended on CCR2, while only a small increase in Ly6C hi monocytes was observed in CCR2 -/- mice. In the lung, significantly higher numbers of Ly6C hi and Ly6C lo monocytes were found in the C57BL/6 mice compared to CCR2 -/- mice. Both wildtype and CCR2-deficient mice developed an inflammatory milieu as shown by cytokine and inos / arg1 mRNA induction in the lung, but with delayed kinetics in CCR2-deficient mice. Histopathology revealed that infiltration of macrophages to the parenchyma, but not into the peribronchial tissue, depended on CCR2. In sum, our data suggest that in Orientia infection, CCR2 drives blood monocytosis and the influx and activation of Ly6C hi and Ly6C lo monocytes into the lung, thereby accelerating bacterial replication and development of interstitial pulmonary inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Petermann, Orfanos, Sellau, Gharaibeh, Lotter, Fleischer and Keller.)

A fracture-related infection (FRI) is a serious complication that can occur after surgical fixation of bone fractures. Affected patients may encounter delayed healing and functional limitations. Although it is well established that Staphylococcus aureus (S. aureus) is the main causative pathogen of an FRI, the pathophysiology of an S. aureus-induced FRI is not well characterised over time. Therefore, an experimental study in mice comparing S. aureus-inoculated and non-inoculated groups was performed that particularly focused on staphylococcal abscess communities (SACs) and host cellular response. C57Bl/6N female mice received a double osteotomy of the femur, which was stabilised using a titanium 6-hole MouseFix locking plate and four screws. Animals were either S. aureus-inoculated or non-inoculated and euthanised between 1 and 28 d post-surgery. Histopathological evaluation showed normal bone healing for non-inoculated mice, whereas inoculated mice had no fracture consolidation and severe osteolysis. Within the bone marrow of inoculated mice, SACs were observed from 7 d, which increased in size and number over time. A fibrin pseudocapsule enclosed the SACs, which were surrounded by many Ly6G+ neutrophils with some Ly6C+ monocytes and F4/80+ macrophages, the majority of which were viable. The abscesses were encapsulated by fibrin(ogen), collagen and myofibroblasts, with regulatory T cells and M2 macrophages at the periphery. Only bone marrow monocytes and neutrophils of inoculated mice displayed functional suppression of T cells, indicative of myeloid-derived suppressor cells. The present study revealed that an FRI in mice is persistent over time and associated with osteolysis, SAC formation and an immunosuppressive environment.

Background: The crucial role of type I interferon (IFN-I, IFN-α/β) is well known to control central nervous system (CNS) neuroinflammation caused by neurotrophic flaviviruses such as Japanese encephalitis virus (JEV) and West Nile virus. However, an in-depth analysis of IFN-I signal-dependent cellular factors that govern CNS-restricted tropism in JEV infection in vivo remains to be elucidated., Methods: Viral dissemination, tissue tropism, and cytokine production were examined in IFN-I signal-competent and -incompetent mice after JEV inoculation in tissues distal from the CNS such as the footpad. Bone marrow (BM) chimeric models were used for defining hematopoietic and tissue-resident cells in viral dissemination and tissue tropism., Results: The paradoxical and interesting finding was that IFN-I signaling was essentially required for CNS neuroinflammation following JEV inoculation in distal footpad tissue. IFN-I signal-competent mice died after a prolonged neurological illness, but IFN-I signal-incompetent mice all succumbed without neurological signs. Rather, IFN-I signal-incompetent mice developed hemorrhage-like disease as evidenced by thrombocytopenia, functional injury of the liver and kidney, increased vascular leakage, and excessive cytokine production. This hemorrhage-like disease was closely associated with quick viral dissemination and impaired IFN-I innate responses before invasion of JEV into the CNS. Using bone marrow (BM) chimeric models, we found that intrinsic IFN-I signaling in tissue-resident cells in peripheral organs played a major role in inducing the hemorrhage-like disease because IFN-I signal-incompetent recipients of BM cells from IFN-I signal-competent mice showed enhanced viral dissemination, uncontrolled cytokine production, and increased vascular leakage. IFN-I signal-deficient hepatocytes and enterocytes were permissive to JEV replication with impaired induction of antiviral IFN-stimulated genes, and neuron cells derived from both IFN-I signal-competent and -incompetent mice were vulnerable to JEV replication. Finally, circulating CD11b + Ly-6C + monocytes infiltrated into the distal tissues inoculated by JEV participated in quick viral dissemination to peripheral organs of IFN-I signal-incompetent mice at an early stage., Conclusion: An IFN-I signal-dependent model is proposed to demonstrate how CD11b + Ly-6C + monocytes are involved in restricting the tissue tropism of JEV to the CNS.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen's ECH&#95;0660 gene, which encodes a phage head-to-tail connector protein, resulted in the rapid clearance of the pathogen in vivo , while aiding in induction of sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH&#95;0659 to ECH&#95;0665, which contained four genes encoding bacterial phage proteins, including the ECH&#95;0660 gene. Assessment of the promoter region upstream of the first gene of the seven genes (ECH&#95;0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation conditions. Furthermore, transcription of the seven genes was significantly higher under zinc and iron starvation conditions for E. chaffeensis carrying a mutation in the ECH&#95;0660 gene compared to the wild-type pathogen. In contrast, for the ECH&#95;0665 gene mutant with the function disruption, transcription from the genes was mostly similar to that of the wild type or was moderately downregulated. Recently, we reported that this mutation caused a minimal impact on the pathogen's in vivo growth, as it persisted similarly to the wild type. The current study is the first to describe how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the phage head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes, including those encoding phage proteins, under zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen's gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo , while aiding in induction of sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the phage head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

Tuberculosis can occur during any stage of Human Immunodeficiency virus 1 (HIV) -infection including times when CD4 + T cell numbers have reconstituted and viral replication suppressed. We have previously shown that CD11b + CD33 + CD14 + HLA-DR -/lo monocytic myeloid-derived suppressor cells (MDSC) persist in HIV-infected individuals on combined anti-retroviral therapy (cART) and with virologic suppression. The response of MDSC to Mycobacterium tuberculosis (Mtb) is not known. In this study, we compared the anti-mycobacterial activity of MDSC isolated from HIV -infected individuals on cART with virologic suppression (HIV MDSC) and HIV-uninfected healthy controls (HIV (-) MDSC). Compared to HIV (-) MDSC, HIV MDSC produced significantly less quantities of anti-mycobacterial cytokines IL-12p70 and TNFα, and reactive oxygen species when cultured with infectious Mtb or Mtb antigens. Furthermore, HIV MDSC showed changes in the Toll-like receptor and IL-27 signaling, including reduced expression of MyD88 and higher levels of IL-27. Neutralizing IL-27 and overexpression of MyD88 synergistically controlled intracellular replication of Mtb in HIV MDSC. These results demonstrate that MDSC in fully suppressed HIV-infected individuals are permissive to Mtb and exhibit downregulated anti-mycobacterial innate immune activity through mechanisms involving IL-27 and TLR signaling. Our findings suggest MDSC as novel mediators of tuberculosis in HIV-Mtb co-infected individuals with virologic suppression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Namdev, Patel, Sparling and Garg.)

Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics. IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease., (Copyright © 2021 Rogan et al.)

Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1β, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c + cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM + myeloid cells and MRP8 + neutrophils, but not CD11c + cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8 + T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR + PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR + LysoDCs) in PP and examine their role in CD8 + T cell immune induction. Stimulation of C5aR + LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8 + T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Decitabine is an approved hypomethylating agent used for treating hematological malignancies. Although decitabine targets altered cells, epidrugs can trigger immunomodulatory effects, reinforcing the hypothesis of immunoregulation in treated patients. We therefore aimed to evaluate the impact of decitabine treatment on the phenotype and functions of monocytes and macrophages, which are pivotal cells of the innate immunity system. In vitro decitabine administration increased bacterial phagocytosis and IL-8 release, but impaired microbicidal activity of monocytes. In addition, during monocyte-to-macrophage differentiation, treatment promoted the M2-like profile, with increased expression of CD206 and ALOX15. Macrophages also demonstrated reduced infection control when exposed to Mycobacterium tuberculosis in vitro. However, cytokine production remained unchanged, indicating an atypical M2 macrophage. Furthermore, when macrophages were cocultured with lymphocytes, decitabine induced a reduction in the release of inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ, maintaining IL-10 production, suggesting that decitabine could potentialize M2 polarization and might be considered as a therapeutic against the exacerbated immune response.

The immune response to mycobacteria is characterized by granuloma formation, which features multinucleated giant cells as a unique macrophage type. We previously found that multinucleated giant cells result from Toll-like receptor-induced DNA damage and cell autonomous cell cycle modifications. However, the giant cell progenitor identity remained unclear. Here, we show that the giant cell-forming potential is a particular trait of monocyte progenitors. Common monocyte progenitors potently produce cytokines in response to mycobacteria and their immune-active molecules. In addition, common monocyte progenitors accumulate cholesterol and lipids, which are prerequisites for giant cell transformation. Inducible monocyte progenitors are so far undescribed circulating common monocyte progenitor descendants with high giant cell-forming potential. Monocyte progenitors are induced in mycobacterial infections and localize to granulomas. Accordingly, they exhibit important immunological functions in mycobacterial infections. Moreover, their signature trait of high cholesterol metabolism may be piggy-backed by mycobacteria to create a permissive niche.

Urocortin (UCN) is a hormone in the hypothalamic-pituitary-adrenal axis that is expressed in various immune cells. However, the function of teleost UCN in the immune system remains unclear. In this study, we cloned the cDNA sequence of UCN from ayu Plecoglossus altivelis (PaUCN). Sequence and phylogenetic tree analyses showed that PaUCN clustered within the fish UCN 1 group and was most related to the rainbow trout (Oncorhynchus mykiss) UCN. PaUCN was expressed in all tested tissues and its expression increased in the liver, spleen, head kidney, and gill upon Vibrio anguillarum infection. Mature PaUCN protein (mPaUCN) treatment affected the phagocytosis and bacterial killing of monocytes/macrophages (MO/MФ). mPaUCN reduced pro-inflammatory cytokine expression in MO/MФ, which was partially mediated via interaction with ayu interleukin-6. mPaUCN reduced bacterial load and increased the survival of V. anguillarum-infected ayu. Overall, UCN as an endocrine factor regulates the immune response of ayu after infection by activating MO/MФ, thus contributing to enhance fish survival., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Six new prenylated indole diketopiperazine alkaloids, asperthrins A-F ( 1 - 6 ), along with eight known analogues ( 7 - 14 ), were isolated from the marine-derived endophytic fungus Aspergillus sp. YJ191021. Their planar structures and absolute configurations were elucidated by HR-ESI-MS, 1D/2D NMR data, and time-dependent density functional theory (TDDFT)/ECD calculation. The isolated compounds were assayed for their inhibition against three agricultural pathogenic fungi, four fish pathogenic bacteria, and two agricultural pathogenic bacteria. Compound 1 exhibited moderate antifungal and antibacterial activities against Vibrio anguillarum, Xanthomonas oryzae pv. Oryzicola , and Rhizoctonia solani with minimal inhibitory concentration (MIC) values of 8, 12.5, and 25 μg/mL, respectively. Furthermore, 1 displayed notable anti-inflammatory activity with IC 50 value of 1.46 ± 0.21 μM in Propionibacterium acnes induced human monocyte cell line (THP-1).

The versatile fish pathogen Edwardsiella tarda is an intracellular pathogen with the ability to invade and replicate in host phagocytes. However, the mechanism mediating the uptake of E. tarda in fish monocytes/macrophages (MO/MΦ) is not yet understood. Generating mudskipper kidney-derived MO/MФ transcriptomic resources from mudskipper challenged by E. tarda is crucial for understanding the molecular mechanisms underlying the mudskipper invasion process. In the present study, a total of 1185 up-regulated and 885 down-regulated differentially expressed genes (DEGs) were identified using RNA-seq. Enrichment and pathway analysis of DEGs revealed the centrality of the phagosome and regulation of actin cytoskeleton pathways in pathogen entry. The progress of phagosome formation was observed by transmission electron microscopy. Eight conserved integrin (ITG) subunit genes, belonging to the phagocytic receptors, were found in the transcriptomic sequence data. Additionally, quantitative real-time PCR showed that the mRNA expressions of most ITG subunit genes were related to the different infection times of E. tarda and the different bacterial pathogens. Further assays demonstrated that phagocytosis of FITC-labeled E. tarda by mudskipper MO/MФ was significantly reduced by the tetrapeptide Asp-Gly-Arg-Ser (RGDS). In summary, phagocytosis is one of the entry pathways into mudskipper MO/MΦ, and RGD-binding ITGs are involved in the phagosome formation process., (Copyright © 2020. Published by Elsevier Ltd.)

Eighteen species of rickettsiae are reported to cause infections in humans. One of these is Rickettsia helvetica, which is endemic in European and Asian countries and transmitted by the tick Ixodes ricinus. Besides fever, it has been demonstrated to cause meningitis and is also associated with perimyocarditis. One of the initial targets for rickettsiae after inoculation by ticks is the macrophage/monocyte. How rickettsiae remain in the macrophages/monocytes before establishing their infection in vascular endothelial cells remains poorly understood. The main aim of the present study was to investigate the impact on and survival of R. helvetica in a human leukemic monocytic cell line, THP-1. Our results show that R. helvetica survives and propagates in the THP-1 cells. The infection in monocytes was followed for seven days by qPCR and for 30 days by TEM, where invasion of the nucleus was also observed as well as double membrane vacuoles containing rickettsiae, a finding suggesting that R. helvetica might induce autophagy at the early stage of infection. Infected monocytes induced TNF-α which may be important in host defence against rickettsial infections and promote cell survival and inhibiting cell death by apoptosis. The present findings illustrate the importance of monocytes to the pathogenesis of rickettsial disease., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis -derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces., Competing Interests: E.R., E.P., N.A., and C.M. are co-inventors on a patent on a tuberculosis vaccine held by the University of Zaragoza and Biofabri. E.R. and E.P. are employees of Biofabri. J.T. is an employee of the TuBerculosis Vaccine Initiative and an advisor to Biofabri. M.G.N. holds a patent on the inhibition of trained immunity with nanobiologics, and a patent on the stimulation of trained immunity with nanobiologics. M.G.N. is also a scientific founder of Trained Therapeutix and Discovery (TTxD). The remaining authors of the Radboud UMC and the authors from the BPRC declare no competing interests., (© 2020 The Author(s).)

To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis -derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies., Competing Interests: E.R., E.P., N.A., and C.M. are co-inventors on a patent-entitled tuberculosis vaccine held by the University of Zaragoza and Biofabri. E.R. and E.P. are employees of Biofabri. J.T. is an employee of TuBerculosis Vaccine Initiative and advisor to Biofabri., (© 2020 The Author(s).)

The human placenta is a dynamic organ that modulates physiological adaptations to pregnancy. To define the immunological signature of the human placenta, we performed unbiased profiling of secreted immune factors from human chorionic villi isolated from placentas at mid and late stages of pregnancy. We show that placental trophoblasts constitutively secrete the inflammasome-associated cytokines IL-1β and IL-18, which is blocked by NLRP3 inflammasome inhibitors and occurs without detectable gasdermin D cleavage. We further show that placenta-derived IL-1β primes monocytes for inflammasome induction to protect against Listeria monocytogenes infection. Last, we show that the human placenta responds to L. monocytogenes infection through additional inflammasome activation and that inhibition of this pathway sensitizes villi to infection. Our results thus identify the inflammasome as an important mechanism by which the human placenta regulates systemic and local immunity during pregnancy to defend against L. monocytogenes infection., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Megli et al.)

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum . Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.

Tuberculosis has been associated with increased risk of atherosclerotic cardiovascular disease. To examine whether mycobacterial infection exacerbates atherosclerosis development in experimental conditions, we infected low-density lipoprotein receptor knockout ( Ldlr -/- ) mice with Mycobacterium bovis Bacille-Calmette-Guérin (BCG), an attenuated strain of the Mycobacterium tuberculosis complex. Twelve-week old male Ldlr -/- mice were infected with BCG (0.3-3.0x10 6 colony-forming units) via the intranasal route. Mice were subsequently fed a western-type diet containing 21% fat and 0.2% cholesterol for up to 16 weeks. Age-matched uninfected Ldlr -/- mice fed with an identical diet served as controls. Atherosclerotic lesions in aorta were examined using Oil Red O staining. Changes induced by BCG infection on the immunophenotyping profile of circulating T lymphocytes and monocytes were assessed using flow cytometry. BCG infection increased atherosclerotic lesions in en face aorta after 8 weeks (plaque ratio; 0.021±0.01 vs. 0.013±0.01; p = 0.011) and 16 weeks (plaque ratio, 0.15±0.13 vs. 0.06±0.02; p = 0.003). No significant differences in plasma cholesterol or triglyceride levels were observed between infected and uninfected mice. Compared to uninfected mice, BCG infection increased systemic CD4/CD8 T cell ratio and the proportion of Ly6C low non-classical monocytes at weeks 8 and 16. Aortic plaque ratios correlated with CD4/CD8 T cell ratios (Spearman's rho = 0.498; p = 0.001) and the proportion of Ly6C low non-classical monocytes (Spearman's rho = 0.629; p < 0.001) at week 16. In conclusion, BCG infection expanded the proportion of CD4 + T cell and Ly6C low monocytes, and aggravated atherosclerosis formation in the aortas of hyperlipidemic Ldlr -/- mice. Our results indicate that mycobacterial infection is capable of enhancing atherosclerosis development., Competing Interests: CF has received research support to the University of Cincinnati from Gilead, Pfizer, BMS, ViiV, Janssen, and Merck. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Huaman, Qualls, Jose, Schmidt, Moussa, Kuhel, Konaniah, Komaravolu, Fichtenbaum, Deepe and Hui.)

Cutibacterium acnes (formerly Propionibacterium acnes ) is a key pathogen involved in the development and progression of acne inflammation. The numerous bioactive properties of wild bitter melon (WBM) leaf extract and their medicinal applications have been recognized for many years. In this study, we examined the suppressive effect of a methanolic extract (ME) of WBM leaf and fractionated components thereof on live C. acnes -induced in vitro and in vivo inflammation. Following methanol extraction of WBM leaves, we confirmed anti-inflammatory properties of ME in C. acnes -treated human THP-1 monocyte and mouse ear edema models. Using a bioassay-monitored isolation approach and a combination of liquid-liquid extraction and column chromatography, the ME was then separated into n -hexane, ethyl acetate, n -butanol and water-soluble fractions. The hexane fraction exerted the most potent anti-inflammatory effect, suppressing C. acnes -induced interleukin-8 (IL-8) production by 36%. The ethanol-soluble fraction (ESF), which was separated from the n -hexane fraction, significantly inhibited C. acne s-induced activation of mitogen-activated protein kinase (MAPK)-mediated cellular IL-8 production. Similarly, the ESF protected against C. acne s-stimulated mouse ear swelling, as measured by ear thickness (20%) and biopsy weight (23%). Twenty-four compounds in the ESF were identified using gas chromatograph-mass spectrum (GC/MS) analysis. Using co-cultures of C. acnes and THP-1 cells, β-ionone, a compound of the ESF, reduced the production of IL-1β and IL-8 up to 40% and 18%, respectively. β-ionone also reduced epidermal microabscess, neutrophilic infiltration and IL-1β expression in mouse ear. We also found evidence of the presence of anti-inflammatory substances in an unfractionated phenolic extract of WBM leaf, and demonstrated that the ESF is a potential anti-inflammatory agent for modulating in vitro and in vivo C. acnes -induced inflammatory responses.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum . E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks. IMPORTANCE The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, human monocytic ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis , is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis -infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis -derived protective antigens that could be used in a candidate vaccine for immunization of humans to counter tick-transmitted ehrlichiosis., (Copyright © 2020 Budachetri et al.)

One new dibenzocycloheptene, validinol ( 1 ), and one butanolide firstly isolated from the natural source, validinolide ( 2 ), together with 17 known compounds were isolated from the stem of Cinnamomum validinerve . Among the isolates, lincomolide A ( 3 ), secosubamolide ( 7 ), and cinnamtannin B1 ( 19 ) exhibited potent inhibition on both superoxide anion generation (IC 50 values of 2.98 ± 0.3 µM, 4.37 ± 0.38 µM, and 2.20 ± 0.3 µM, respectively) and elastase release (IC 50 values of 3.96 ± 0.31 µM, 3.04 ± 0.23 µM, and 4.64 ± 0.71 µM, respectively) by human neutrophils. In addition, isophilippinolide A ( 6 ), secosubamolide ( 7 ), and cinnamtannin B1 ( 19 ) showed bacteriostatic effects against Propionibacterium acnes in in vitro study, with minimal inhibitory concentration (MIC) values at 16 μg/mL, 16 μg/mL, and 500 μg/mL, respectively. Further investigations using the in vivo ear P. acnes infection model showed that the intraperitoneal administration of the major component cinnamtannin B1 ( 19 ) reduced immune cell infiltration and pro-inflammatory cytokines TNF-α and IL-6 at the infection sites. The results demonstrated the potential of cinnamtannin B1 ( 19 ) for acne therapy. In summary, these results demonstrated the anti-inflammatory potentials of Formosan C. validinerve during bacterial infections.

Leptospirosis remains a significant human health issue due to its systemic complications. Therefore, biomarkers that are more effective are urgently needed for the early diagnosis of leptospirosis. MicroRNAs (miRNAs) are evolutionarily conserved regulatory RNAs that have shown the potential to be used as biomarkers for diagnosis, prognosis, and therapy of infectious diseases. In this study, we performed an unbiased screen using the miRNome miRNA array to identify circulating miRNAs with the potential to serve as authentic biomarkers for early diagnosis of leptospirosis. Because leptospiral lipopolysaccharide (LPS) is the predominant leptospiral antigen and plays a vital role in immunological and biological activities, we used LPS treated and untreated in vitro (THP1 cells) and in vivo (BALB/c mice) surrogate models to identify the LPS-specific miRNAs. Differential expression analysis revealed 18 miRNAs to be associated strongly with LPS stimulation in THP1 cells. Of these, three (miR-let-7b-5p, miR-144-3p, and miR-21-5p) were observed to be present at increased levels in vivo The identified miRNAs were validated for their biomarker potential using serum samples from leptospirosis-negative patients and patients with confirmed cases of leptospirosis. Identified miRNAs were able to discriminate the acute leptospiral infection from other febrile diseases with a test sensitivity and specificity of 93.2% and 88.19%, respectively. Gene functional enrichment and protein-protein interaction (PPI) network analysis revealed that the identified miRNAs play important roles in disease signal transduction, signaling by interleukins, the stress-activated protein kinase signaling cascade, the mitogen-activated protein kinase (MAPK) signaling pathway, and the cellular response to a transforming growth factor β (TGF-β) stimulus with a notable interconnection between these biological processes. IMPORTANCE Here, we used miRNAs that are differentially regulated by the LPS/TLR2 immune axis to devise a miRNA-based diagnosis for leptospirosis. The study established the role of the circulating stable miRNAs (miR-21-5p, miR-144-3p, and miR-let-7b-5p) as an early diagnostic marker for leptospirosis. These miRNAs can be used to diagnose acute leptospirosis and also to differentiate leptospiral infection from other bacterial and spirochetal infections, as proved by the use of human clinical samples. Thus, our findings indicate that miRNAs can play a crucial role in the diagnosis of infectious diseases, like leptospirosis, that are generally misdiagnosed., (Copyright © 2020 Akino Mercy et al.)

Tissue damage triggers a rapid and robust inflammatory response in order to clear and repair a wound. Remarkably, many of the cell biology features that underlie the ability of leukocytes to home in to sites of injury and to fight infection-most of which are topics of intensive current research-were originally observed in various weird and wonderful translucent organisms over a century ago by Elie Metchnikoff, the "father of innate immunity," who is credited with discovering phagocytes in 1882. In this review, we use Metchnikoff's seminal lectures as a starting point to discuss the tremendous variety of cell biology features that underpin the function of these multitasking immune cells. Some of these are shared by other cell types (including aspects of motility, membrane trafficking, cell division, and death), but others are more unique features of innate immune cells, enabling them to fulfill their specialized functions, such as encapsulation of invading pathogens, cell-cell fusion in response to foreign bodies, and their self-sacrifice as occurs during NETosis., (© 2020 Weavers and Martin.)

Type-I interferon (IFN-I) cytokines are produced by immune cells in response to microbial infections, cancer and autoimmune diseases, and subsequently, trigger cytoprotective and antiviral responses through the activation of IFN-I stimulated genes (ISGs). The ability of intestinal microbiota to modulate innate immune responses is well known, but the mechanisms underlying such responses remain elusive. Here we report that the intracellular sensors stimulator of IFN genes (STING) and mitochondrial antiviral signaling (MAVS) are essential for the production of IFN-I in response to lactic acid bacteria (LAB), common gut commensal bacteria with beneficial properties. Using human macrophage cells we show that LAB strains that potently activate the inflammatory transcription factor NF-κB are poor inducers of IFN-I and conversely, those triggering significant amounts of IFN-I fail to activate NF-κB. This IFN-I response is also observed in human primary macrophages, which modulate CD64 and CD40 upon challenge with IFN-I-inducing LAB. Mechanistically, IFN-I inducers interact more intimately with phagocytes as compared to NF-κB-inducers, and fail to activate IFN-I in the presence of phagocytosis inhibitors. These bacteria are then sensed intracellularly by the cytoplasmic sensors STING and, to a lesser extent, MAVS. Accordingly, macrophages deficient for STING showed dramatically reduced phosphorylation of TANK-binding kinase (TBK)-1 and IFN-I activation, which resulted in lower expression of ISGs. Our findings demonstrate a major role for intracellular sensing and STING in the production of IFN-I by beneficial bacteria and the existence of bacteria-specific immune signatures, which can be exploited to promote cytoprotective responses and prevent overreactive NF-κB-dependent inflammation in the gut.

During tuberculosis, lung myeloid cells have two opposing roles: they are an intracellular niche occupied by Mycobacterium tuberculosis, and they restrict bacterial replication. Lung myeloid cells from mice infected with yellow-fluorescent protein expressing M. tuberculosis were analyzed by flow cytometry and transcriptional profiling to identify the cell types infected and their response to infection. CD14, CD38, and Abca1 were expressed more highly by infected alveolar macrophages and CD11cHi monocyte-derived cells compared to uninfected cells. CD14, CD38, and Abca1 "triple positive" (TP) cells had not only the highest infection rates and bacterial loads, but also a strong interferon-γ signature and nitric oxide synthetase-2 production indicating recognition by T cells. Despite evidence of T cell recognition and appropriate activation, these TP macrophages are a cellular compartment occupied by M. tuberculosis long-term. Defining the niche where M. tuberculosis resists elimination promises to provide insight into why inducing sterilizing immunity is a formidable challenge., Competing Interests: The authors have declared that no competing interests exist.

IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing, and pathogen immunity. IRGM dysfunction is linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction., Competing Interests: I have read the journal's policy and have the following competing interests: B.A.V. is the Children with Intestinal and Liver Disorders (C.H.I.L.D.) Foundation Chair in Pediatric Gastroenterology.

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis . The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD + and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT ; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (P mbcA ) in a variety of stress conditions, including in vivo in mice and in macrophages.