Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection.

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/-ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/-ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.

Role of Dectin-2 in the Phagocytosis of Cryptococcus neoformans by Dendritic Cells.

The cell walls and capsules of Cryptococcus neoformans, a yeast-type fungal pathogen, are rich in polysaccharides. Dectin-2 is a C-type lectin receptor (CLR) that recognizes high-mannose polysaccharides. Previously, we demonstrated that Dectin-2 is involved in cytokine production by bone marrow-derived dendritic cells (BM-DCs) in response to stimulation with C. neoformans. In the present study, we analyzed the role of Dectin-2 in the phagocytosis of C. neoformans by BM-DCs. The engulfment of this fungus by BM-DCs was significantly decreased in mice lacking Dectin-2 (Dectin-2 knockout [Dectin-2KO]) or caspase recruitment domain-containing protein 9 (CARD9KO), a common adapter molecule that delivers signals triggered by CLRs, compared to wild-type (WT) mice. Phagocytosis was likewise inhibited, to a similar degree, by the inhibition of Syk, a signaling molecule involved in CLR-triggered activation. A PI3K inhibitor, in contrast, completely abrogated the phagocytosis of C. neoformans. Actin polymerization, i.e., conformational changes in cytoskeletons detected at sites of contact with C. neoformans, was also decreased in BM-DCs of Dectin-2KO and CARD9KO mice. Finally, the engulfment of C. neoformans by macrophages was significantly decreased in the lungs of Dectin-2KO mice compared to WT mice. These results suggest that Dectin-2 may play an important role in the actin polymerization and phagocytosis of C. neoformans by DCs, possibly through signaling via CARD9 and a signaling pathway mediated by Syk and PI3K.

α-Hemolysin suppresses osteogenesis by inducing lipid rafts accumulation in bone marrow stromal cells.

α-hemolysin (Hla) is considered an essential virulent factor for Staphylococcus aureus (S. aureus) toxicity, the mechanism by which Hla affect bone metabolism is poorly understood. In this study, 2-month-old C57BL/6 mice were treated with Hla (40 µg/kg, i.p.) or S. aureus (1 × 106 CFU/ml, 100 µl, i.v.) with the presence or absence of methyl-ß-cyclodextrin (MßCD) (300 mg/kg, i.p.). MicroCT analysis showed progressive bone loss from week 2 to week 4 after Hla treatment, accompanied by a decreased osteoblasts and increased osteoclasts in femoral metaphysis in mice. Further, Hla stimulated the expression of Caveolin-1 in vivo and in vitro, activated lipid rafts accumulation in cell membrane of bone marrow stromal cells (BMSCs), and suppressed osteogenesis of BMSCs. Destruction of lipid rafts with MßCD or inhibition of Caveolin-1 with Daidzein blocked the detrimental effect of Hla on osteogenesis of BMSCs. Importantly, treating mice with MßCD rescued the loss of osteoblasts and increased osteoclastogenesis induced by Hla as well as the bone loss induced by S. aureus infection. Together, we demonstrate that Hla induces bone destruction directly by suppressing osteogenesis and indirectly by stimulating osteoclastogenesis, and that lipid rafts may mediate the detrimental effect of Hla and S. aureus on osteogenesis and bone formation.

Microbial sensing by haematopoietic stem and progenitor cells: Vigilance against infections and immune education of myeloid cells.

Bone marrow haematopoietic stem and progenitor cells (HSPCs) express pattern recognition receptors such as Toll-like receptors (TLRs) to sense microbial products and activation of these innate immune receptors induces cytokine expression and redirects bone marrow haematopoiesis towards the increased production of myeloid cells. Secreted cytokines by HSPCs in response to TLR ligands can act in an autocrine or paracrine manner to regulate haematopoiesis. Moreover, tonic activation of HSPCs by microbiota-derived compounds might educate HSPCs to produce superior myeloid cells equipped with innate memory responses to combat pathogens. While haematopoietic stem cell activation through TLRs meets the increased demand for blood leucocytes to protect the host against infection, persistent exposure to inflammatory cytokines or microbial products might impair their function and even induce malignant transformation. This review highlights the potential outcomes of HSPCs in response to TLR ligands.

The Ubiquitin-Modifying Enzyme A20 Terminates C-Type Lectin Receptor Signals and Is a Suppressor of Host Defense against Systemic Fungal Infection.

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.

Spleen Tyrosine Kinase Is a Critical Regulator of Neutrophil Responses to Candida Species.

Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to Candida species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens Candida albicans, Candida glabrata, and Candida auris Neutrophil responses to Candida species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to Candida species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics.IMPORTANCE Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen Candida The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic Candida species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders.

Antibody, but not B-cell-dependent antigen presentation, plays an essential role in preventing Chlamydia systemic dissemination in mice.

The obligate intracellular bacterium Chlamydia trachomatis causes the most prevalent bacterial sexually transmitted infection worldwide. CD4 T cells play a central role in the protective immunity against Chlamydia female reproductive tract (FRT) infection, while B cells are thought to be dispensable for resolution of primary Chlamydia infection in mouse models. We recently reported an unexpected requirement of B cells in local Chlamydia-specific CD4 T-cell priming and bacterial containment within the FRT. Here, we sought to tackle the precise effector function of B cells during Chlamydia primary infection. Using mixed bone marrow chimeras that lack B-cell-dependent Ag presentation (MHCIIB-/- ) or devoid of circulating antibodies (AID-/- × µS-/- ), we show that Chlamydia-specific CD4 T-cell expansion does not rely on Ag presentation by B cells. Importantly, we demonstrate that antibody, but not B-cell-dependent Ag presentation, is required for preventing systemic bacterial dissemination following Chlamydia FRT infection.

Initiation of Post-Primary Tuberculosis of the Lungs: Exploring the Secret Role of Bone Marrow Derived Stem Cells.

Mycobacterium tuberculosis (Mtb), the causative organism of pulmonary tuberculosis (PTB) now infects more than half of the world population. The efficient transmission strategy of the pathogen includes first remaining dormant inside the infected host, next undergoing reactivation to cause post-primary tuberculosis of the lungs (PPTBL) and then transmit via aerosol to the community. In this review, we are exploring recent findings on the role of bone marrow (BM) stem cell niche in Mtb dormancy and reactivation that may underlie the mechanisms of PPTBL development. We suggest that pathogen's interaction with the stem cell niche may be relevant in potential inflammation induced PPTBL reactivation, which need significant research attention for the future development of novel preventive and therapeutic strategies for PPTBL, especially in a post COVID-19 pandemic world. Finally, we put forward potential animal models to study the stem cell basis of Mtb dormancy and reactivation.

Interaction of Cutibacterium acnes with human bone marrow derived mesenchymal stem cells: a step toward understanding bone implant- associated infection development.

Crosstalk between mesenchymal stem cells (MSCs) and bacteria plays an important role in regulating the regenerative capacities of MSCs, fighting infections, modulating immune responses and maintaining tissue homeostasis. Commensal Cutibacterium acnes (C. acnes) bacterium becomes an opportunistic pathogen causing implant-associated infections. Herein, we examined MSCs/C. acnes interaction and analysed the subsequent bacteria and MSCs behaviours following infection. Human bone marrow derived MSCs were infected by two clinical and one laboratory C. acnes strains. Following 3h of interaction, all bacterial strains were able to invade MSCs. Viable intracellular bacteria acquired virulence factors by increasing biofilm formation and/or by affecting macrophage phagocytosis. Although the direct and indirect (through neutrophil stimulation) antibacterial effects of the MSCs secretome were not enhanced following C. acnes infection, ELISA analysis revealed that C. acnes clinical strains are able to license MSCs to become immunosuppressive cell-like by increasing the secretion of IL-6, IL-8, PGE-2, VEGF, TGF-ß and HGF. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections. STATEMENT OF SIGNIFICANCE: The originality of this work relies on the study of relationship between human bone marrow derived mesenchymal stem cells (MSCs) phenotype and C. acnes clinical strains virulence following cell infection. Our major results showed that C. acnes are able to invade MSCs, inducing a transition of commensal to an opportunistic pathogen behaviour. Although the direct and indirect antibacterial effects were not enhanced following C. acnes infection, secretome analysis revealed that C. acnes clinical strains were able to license MSCs to become immunosuppressive and anti-fibrotic cell-like. These results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during associated implant infections.

Two-staged time-dependent materials for the prevention of implant-related infections.

Infection is a main cause of implant failure. Early implant-related infections often occur in the first 4 weeks post-operation. Inhibiting bacterial adhesion and biofilm formation at the early stage and promoting subsequent implant osseointegration are important for implant success. Our previous studies demonstrated that dimethylaminododecyl methacrylate (DMADDM) provided dental materials with antibacterial effects. In the present study, DMADDM and hydroxyapatite (HA) are loaded on to the titanium (Ti) surface via poly dopamine (PDA) self-polymerization. This local DMADDM-delivery Ti is referred as Ti-PHD. Here we report the two-staged capability of Ti-PHD: (1) in the first stage, releasing DMADDM during the high-infection-risk initial period post-implantation for 4 weeks; (2) then in the second stage, enhancing osteogenesis and promoting osseointegration. Ti-PHD has a porous surface with higher average roughness and greater hydrophilicity than pure Ti. Its biocompatibility is verified in vitro and in vivo. During the first 4 weeks of release, both DMADDM remaining on Ti surface and DMADDM released into the soaking medium greatly reduced the adherence and growth of pathogens. This is further confirmed by the prevention of bone destruction in a rat osteomyelitis model. After releasing DMADDM for 4 weeks, Ti-PHD promotes osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and new bone formation around the implants in vivo. This article represents the first report on the two-staged, time-dependent antibacterial and osteogenesis effects of Ti-PHD, demonstrating its potential for clinical applications to inhibit implant-associated infections. STATEMENT OF SIGNIFICANCE: The present study develops a two-staged time-dependent system for local dimethylaminododecyl methacrylate (DMADDM) delivery via Ti implant (referred to as Ti-PHD). DMADDM and hydroxyapatite (HA) are loaded on to the Ti surface with poly dopamine (PDA). Ti-PHD can release DMADDM during the high-risk period of infection in the first stage, and then promote osseointegration and new bone formation in the second stage. This bioactive and therapeutic Ti is promising to inhibit infections and enhance implant success.

Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond.

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.

A rare case of disseminated penicilliosis - first of its kind from North India.

Penicilliosis is a rare opportunistic fungal infection caused by Talaromyces marneffei, especially in the HIV-infected patients. The untreated disease is highly fatal. The infection is endemic in Southeast Asia and Northeast India. The present case is the first case of disseminated penicilliosis from North India and Delhi in a 31-year-old male, recently diagnosed with HIV. This case highlights the importance of considering an unusual organism as the cause of disseminated disease in the nonendemic area.

The role of myeloid-derived suppressor cells in chronic infectious diseases and the current methodology available for their study.

An effective pathogen has the ability to evade the immune response. The strategies used to achieve this may be based on the direct action of virulence factors or on the induction of host factors. Myeloid-derived suppressor cells (MDSCs) are immune cells with an incredible ability to suppress the inflammatory response, which makes them excellent targets to be exploited by pathogenic bacteria, viruses, or parasites. In this review, we describe the origin and suppressive mechanisms of MDSCs, as well as their role in chronic bacterial, viral, and parasitic infections, where their expansion seems to be essential in the chronicity of the disease. We also analyze the disadvantages of current MDSC depletion strategies and the different in vitro generation methods, which can be useful tools for the deeper study of these cells in the context of microbial infections.

Comparison of Antibacterial and Immunological Properties of Mesenchymal Stem/Stromal Cells from Equine Bone Marrow, Endometrium, and Adipose Tissue.

Equine mesenchymal stem/stromal cells (MSCs) are multipotent cells that are widely used for treatment of musculoskeletal injuries, and there is significant interest in expanding their application to nonorthopedic conditions. MSCs possess antibacterial and immunomodulatory properties that may be relevant for combating infection; however, comparative studies using MSCs from different origins have not been carried out in the horse, and this was the focus of this study. Our results showed that MSC-conditioned media attenuated the growth of Escherichia coli, and that this effect was, on average, more pronounced for endometrium (EM)-derived and adipose tissue (AT)-derived MSCs than for bone marrow (BM)-derived MSCs. In addition, the antimicrobial lipocalin-2 was expressed at mean higher levels in EM-MSCs than in AT-MSCs and BM-MSCs, and the bacterial component lipopolysaccharide (LPS) stimulated its production by all three MSC types. We also showed that MSCs express interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, chemokine ligand-5, and Toll-like receptor 4, and that, in general, these cytokines were induced in all cell types by LPS. Low expression levels of the macrophage marker colony-stimulating factor 1 receptor were detected in BM-MSCs and EM-MSCs but not in AT-MSCs. Altogether, these findings suggest that equine MSCs from EM, AT, and BM have both direct and indirect antimicrobial properties that may vary between MSCs from different origins and could be exploited toward improvement of regenerative therapies for horses.

Hypoxia induces senescence of bone marrow mesenchymal stem cells via altered gut microbiota.

Systemic chronic hypoxia is a feature of many diseases and may influence the communication between bone marrow (BM) and gut microbiota. Here we analyse patients with cyanotic congenital heart disease (CCHD) who are experiencing chronic hypoxia and characterize the association between bone marrow mesenchymal stem cells (BMSCs) and gut microbiome under systemic hypoxia. We observe premature senescence of BMSCs and abnormal D-galactose accumulation in patients with CCHD. The hypoxia that these patients experience results in an altered diversity of gut microbial communities, with a remarkable decrease in the number of Lactobacilli and a noticeable reduction in the amount of enzyme-degraded D-galactose. Replenishing chronic hypoxic rats with Lactobacillus reduced the accumulation of D-galactose and restored the deficient BMSCs. Together, our findings show that chronic hypoxia predisposes BMSCs to premature senescence, which may be due to gut dysbiosis and thus induced D-galactose accumulation.

Disseminated histoplasmosis: case report and review of the literature.

Case report We report the case of a young Cameroonian woman who presented with cough, hyperthermia, weight loss, pancytopenia, and hepatosplenomegaly. A positive HIV serology was discovered and a chest radiography revealed a 'miliary pattern'. Bone marrow aspiration pointed out yeast inclusions within macrophages. Given the morphological aspect, the clinical presentation and immunosuppression, histoplasmosis was retained as a working hypothesis. Antiretroviral and amphotericin B treatments were promptly initiated. Review Given the immigration wave that Europe is currently experiencing, we think it is important to share experience and knowledge, especially in non-endemic areas such as Europe, where clinicians are not used to face this disease. Histoplasmosis is due to Histoplasma capsulatum var. capsulatum, a dimorphic fungus. Infection occurs by inhaling spores contained in soils contaminated by bat or bird droppings. The clinical presentation depends on the immune status of the host and the importance of inoculum, varying from asymptomatic to disseminated forms. AIDS patients are particularly susceptible to develop a severe disease. Antigen detection, molecular biology techniques, and microscopic examination are used to make a rapid diagnosis. However, antigen detection is not available in Europe and diagnosis needs a strong clinical suspicion in non-endemic areas. Because of suggestive imagery, clinicians might focus on tuberculosis. Our case illustrates the need for clinicians to take histoplasmosis in the differential diagnosis, depending on the context and the patient's past history.

Type I interferon enhances necroptosis of Salmonella Typhimurium-infected macrophages by impairing antioxidative stress responses.

Salmonella enterica serovar Typhimurium exploits the host's type I interferon (IFN-I) response to induce receptor-interacting protein (RIP) kinase-mediated necroptosis in macrophages. However, the events that drive necroptosis execution downstream of IFN-I and RIP signaling remain elusive. In this study, we demonstrate that S Typhimurium infection causes IFN-I-mediated up-regulation of the mitochondrial phosphatase Pgam5 through RIP3. Pgam5 subsequently interacts with Nrf2, which sequesters Nrf2 in the cytosol, thereby repressing the transcription of Nrf2-dependent antioxidative genes. The impaired ability to respond to S Typhimurium-induced oxidative stress results in reactive oxygen species-mediated mitochondrial damage, energy depletion, transient induction of autophagy, and autophagic degradation of p62. Reduced p62 levels impair interaction of p62 with Keap1, which further decreases Nrf2 function and antioxidative responses to S Typhimurium infection, eventually leading to cell death. Collectively, we identify impaired Nrf2-dependent redox homeostasis as an important mechanism that promotes cell death downstream of IFN-I and RIP3 signaling in S Typhimurium-infected macrophages.

Mouse Bone Marrow Sca-1+ CD44+ Mesenchymal Stem Cells Kill Avirulent Mycobacteria but Not Mycobacterium tuberculosis through Modulation of Cathelicidin Expression via the p38 Mitogen-Activated Protein Kinase-Dependent Pathway.

Mycobacterium tuberculosis primarily infects lung macrophages. However, a recent study showed that M. tuberculosis also infects and persists in a dormant form inside bone marrow mesenchymal stem cells (BM-MSCs) even after successful antibiotic therapy. However, the mechanism(s) by which M. tuberculosis survives in BM-MSCs is still not known. Like macrophages, BM-MSCs do not contain a well-defined endocytic pathway, which is known to play a central role in the clearance of internalized mycobacteria. Here, we studied the fate of virulent and avirulent mycobacteria in Sca-1+ CD44+ BM-MSCs. We found that BM-MSCs were able to kill avirulent Mycobacterium smegmatis and Mycobacterium bovis BCG but not the pathogenic species M. tuberculosis Further mechanistic studies revealed that pathogenic M. tuberculosis dampens the antibacterial response of BM-MSCs by downregulating the expression of the cationic antimicrobial peptide cathelicidin. In contrast, avirulent mycobacteria were effectively killed by inducing the Toll-like receptor 2/4 (TLR2/4) pathway-dependent expression of cathelicidin, while small interfering RNA (siRNA)-mediated cathelicidin silencing increased the survival of M. bovis BCG in BM-MSCs. We also showed that M. bovis BCG infection caused increased expression levels of MyD88, phospho-interleukin-1 receptor-associated kinase 4 (pIRAK-4), and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Further downstream investigations demonstrated that IRAK-4-p38 activation increased the nuclear translocation of NF-κB, which subsequently induced the expression of cathelicidin and the cytokine interleukin-1ß (IL-1ß), resulting in the decreased survival of M. bovis BCG. On the other hand, inhibition of TLR2/4, pIRAK-4, p38, and NF-κB nuclear translocation decreased cathelicidin and IL-1ß expression levels and therefore increased the survival of avirulent mycobacteria. This is the first report that demonstrates that virulent mycobacteria manipulate the TLR2/4-MyD88-IRAK-4-p38-NF-κB-Camp-IL-1ß pathway to survive inside bone marrow stem cells.