Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases.

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein-protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.

DNA from virulent M. tuberculosis induces TNF-α production and autophagy in M1 polarized macrophages.

The macrophage innate immune response is outlined through recognition of the components of Mycobacterium tuberculosis. DNA of M. tuberculosis (MtbDNA) is recognized by macrophages, but the implications of this recognition are poorly characterized. Stimulation of murine macrophages with MtbDNA induces autophagy, a process that promotes elimination of intracellular pathogens. However, it remains unknown whether this or other phenomena also occur in human cells. In this work, we studied the innate response profiles of human macrophages after stimulation with DNA from virulent M. tuberculosis H37Rv. Human monocyte-derived macrophages were polarized into M1 and M2 phenotypes and stimulated with MtbDNA. The plasma membrane markers of the phenotype, production of TNF-α, and induction of autophagy were evaluated. Our results indicate that MtbDNA induced phenotypical changes, the significant production of TNF-α, and autophagy confirmed by the augmented expression of immunity related GTPase M (IRGM) and autophagy related ATG16L1 genes in M1 macrophages, whereas M2 macrophages exhibited limited responses. In addition, MtbDNA activation was TLR-9-dependent. Although TLR-9 expression was similar between M1 and M2 macrophages, only M1 macrophages were fully responsive to MtbDNA. In conclusion, MtbDNA recognition enhanced the antimicrobial mechanisms of M1 macrophages.

Human macrophages chronically exposed to LPS can be reactivated by stimulation with MDP to acquire an antimicrobial phenotype.

Macrophages are important in host defense and can differentiate into functionally distinct subsets named classically (M1) or alternatively (M2) activated. In several inflammatory disorders, macrophages become tolerized to prevent deleterious consequences. This tolerization reduces the ability of macrophages to respond to bacterial components (e.g., LPS) maintaining a low level of inflammation but compromising the ability of macrophages to mount an effective immune response during subsequent pathogen encounters. In this study, we aimed to reactivate human monocyte-derived macrophages chronically exposed to LPS by re-stimulation with muramyl dipeptide (MDP). We observed an undefined profile of cell surface marker expression during endotoxin tolerance and absence of TNFα production. Stimulating macrophages chronically exposed to LPS with LPS+MDP restored TNFα, production together with an increased production of IL1, IL6, IFNγ, IL4, IL5 and IL10. These results suggest that macrophages chronically exposed to LPS possess a mixed M1-M2 phenotype with sufficient antimicrobial and homeostatic potential.

High glucose induces O-GlcNAc glycosylation of the vitamin D receptor (VDR) in THP1 cells and in human macrophages derived from monocytes.

Chronic hyperglycemia increases the carbon flux through the hexosamine pathway, allowing the accumulation of UDP-GlcNAc. UDP-GlcNAc is the sugar donor for the enzyme-mediated protein glycosylation event known as OGlcNAcylation. This posttranslational modification targets several transcription factors implicated in glucose toxicity, insulin resistance, and diabetes. Vitamin D plays an important role in glucose homeostasis and insulin secretion through transcriptional mechanisms mediated by its receptor (VDR). Vitamin D deficiency has been associated with higher susceptibility to bacterial diseases in diabetic patients. However, it has not been explored whether VDR is subject to OGlcNAcylation or whether high glucose affects its transcriptional or biological activities. The aim of this study was to evaluate the effect of hyperglycemia on VDR OGlcNAcylation and its effects on vitamin D-mediated transcription. We predicted potential OGlcNAcylation sites using free software. Our results showed that hyperglycemia (30 mM) induces the OGlcNAcylation of VDR in THP1 cells and in human macrophages derived from monocytes (MDM). This condition did not hamper the vitamin D-dependent activation of LL-37 gene expression, and even did not impair the macrophage bactericidal activity. Our study provides new insight into vitamin D receptor posttranslational modification that may have relevance on the physiological responses of long-term hyperglycemia.

Bactericidal Permeability-Increasing Protein (BPI) Inhibits Mycobacterium tuberculosis Growth.

Bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils, eosinophils, fibroblasts, and macrophages with antibacterial anti-inflammatory properties. In the context of Gram-negative infection, BPI kills bacteria, neutralizes the endotoxic activity of lipopolysaccharides (LPSs), and, thus, avoids immune hyperactivation. Interestingly, BPI increases in patients with Gram-positive meningitis, interacts with lipopeptides and lipoteichoic acids of Gram-positive bacteria, and significantly enhances the immune response in peripheral blood mononuclear cells. We evaluated the antimycobacterial and immunoregulatory properties of BPI in human macrophages infected with Mycobacterium tuberculosis. Our results showed that recombinant BPI entered macrophages, significantly reduced the intracellular growth of M. tuberculosis, and inhibited the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, BPI decreased bacterial growth directly in vitro. These data suggest that BPI has direct and indirect bactericidal effects inhibiting bacterial growth and potentiating the immune response in human macrophages and support that this new protein's broad-spectrum antibacterial activity has the potential for fighting tuberculosis.

Humoral and cellular responses to a non-adjuvanted monovalent H1N1 pandemic influenza vaccine in hospital employees.

BACKGROUND: The efficacy of the H1N1 influenza vaccine relies on the induction of both humoral and cellular responses. This study evaluated the humoral and cellular responses to a monovalent non-adjuvanted pandemic influenza A/H1N1 vaccine in occupationally exposed subjects who were previously vaccinated with a seasonal vaccine. METHODS: Sixty healthy workers from a respiratory disease hospital were recruited. Sera and peripheral blood mononuclear cells (PBMCs) were obtained prior to and 1 month after vaccination with a non-adjuvanted monovalent 2009 H1N1 vaccine (Influenza A (H1N1) 2009 Monovalent Vaccine Panenza, Sanofi Pasteur). Antibody titers against the pandemic A/H1N1 influenza virus were measured via hemagglutination inhibition (HI) and microneutralization assays. Antibodies against the seasonal HA1 were assessed by ELISA. The frequency of IFN-γ-producing cells as well as CD4+ and CD8+ T cell proliferation specific to the pandemic virus A/H1N peptides, seasonal H1N1 peptides and seasonal H3N2 peptides were assessed using ELISPOT and flow cytometry. RESULTS: At baseline, 6.7% of the subjects had seroprotective antibody titers. The seroconversion rate was 48.3%, and the seroprotection rate was 66.7%. The geometric mean titers (GMTs) were significantly increased (from 6.8 to 64.9, p < 0.05). Forty-nine percent of the subjects had basal levels of specific IFN-γ-producing T cells to the pandemic A/H1N1 peptides that were unchanged post-vaccination. CD4+ T cell proliferation in response to specific pandemic A/H1N1 virus peptides was also unchanged; in contrast, the antigen-specific proliferation of CD8+ T cells significantly increased post-vaccination. CONCLUSION: Our results indicate that a cellular immune response that is cross-reactive to pandemic influenza antigens may be present in populations exposed to the circulating seasonal influenza virus prior to pandemic or seasonal vaccination. Additionally, we found that the pandemic vaccine induced a significant increase in CD8+ T cell proliferation.

Exploring COX-2 inhibitors in tuberculosis: A whole-blood model approach for immune response and adjunt therapy evaluation.

Pulmonary tuberculosis (TB) inflammation is an underestimated disease complication which anti-inflammatory drugs may alleviate. This study explored the potential use of the COX-2 inhibitors acetylsalicylic acid (ASA) and celecoxib in 12 TB patients and 12 healthy controls using a whole-blood ex vivo model where TNFα, PGE2, and LTB4 plasma levels were quantitated by ELISA; we also measured COX-2, 5-LOX, 12-LOX, and 15-LOX gene expression. We observed a significant TNFα production in response to stimulation with LPS or M. tuberculosis (Mtb). Celecoxib, but not ASA, reduced TNFα and PGE2 production, while increasing LTB4 in patients after infection with Mtb. Gene expression of COX-2 and 5-LOX was higher in controls, while 12-LOX was significantly higher in patients. 15-LOX expression was similar in both groups. We concluded that COX-2 inhibitors downregulate inflammation after Mtb infection, and our methodology offers a straightforward time-efficient approach for evaluating different drugs in this context. Further research is warranted to elucidate the underlying mechanisms and assess the potential clinical benefit.

Serum pro-inflammatory biomarkers associated with improvement in quality of life in pulmonary tuberculosis.

Introduction: Pulmonary dysfunction is an underestimated complication in tuberculosis (TB) infection, affecting quality of life (QoL). Although respiratory function tests objectively reflect lung disturbances in a specific moment, predictors of illness severity at the time of diagnosis are still lacking. Methods: We measured serum pro-inflammatory cytokines (TNF-α and IL-8), eicosanoids (PGE2, LTB4, RvD1, Mar1, and LXA4), a marker of tissue damage (cell-free nucleosomes), and indicators of redox status (malonaldehyde, 8-isoprostane, total oxidants, and antioxidants), as well as a score of radiological abnormalities (SRA) and a QoL questionnaire, in 25 patients with pulmonary TB at the time of diagnosis (t0) and two months after the initiation of treatment (t2). Results: We found higher antioxidant levels in the patients with the worst QoL at t0, and all the indicators of the prooxidant state were significantly reduced at t2, while the total antioxidant levels increased. LTB4, a pro-inflammatory eicosanoid, was diminished at t2, while all the pro-resolutory lipids decreased substantially. Significant correlations between the SRA and the QoL scores were observed, the latter showing a substantial reduction at t2, ranking it as a reliable tool for monitoring disease evolution during TB treatment. Discussion: These results suggest that evaluating a combination of these markers might be a valuable predictor of QoL improvement and a treatment response indicator; in particular, the oxidation metabolites and eicosanoid ratios could also be proposed as a future target for adjuvant therapies to reduce inflammation-associated lung injury in TB disease.

Reduced IL-8 Secretion by NOD-like and Toll-like Receptors in Blood Cells from COVID-19 Patients.

Severe inflammatory responses are associated with the misbalance of innate and adaptive immunity. TLRs, NLRs, and cytokine receptors play an important role in pathogen sensing and intracellular control, which remains unclear in COVID-19. This study aimed to evaluate IL-8 production in blood cells from COVID-19 patients in a two-week follow-up evaluation. Blood samples were taken at admission (t1) and after 14 days of hospitalization (t2). The functionality of TLR2, TLR4, TLR7/8, TLR9, NOD1, and NOD2 innate receptors and IL-12 and IFN-γ cytokine receptors was evaluated by whole blood stimulation with specific synthetic receptor agonists through the quantification of IL-8, TNF-α, or IFN-γ. At admission, ligand-dependent IL-8 secretion was 6.4, 13, and 2.5 times lower for TLR2, TLR4, and endosomal TLR7/8 receptors, respectively, in patients than in healthy controls. Additionally, IL-12 receptor-induced IFN-γ secretion was lower in COVID-19 patients than in healthy subjects. We evaluated the same parameters after 14 days and observed significantly higher responses for TLR2, TLR4, TLR7/8, TLR9, and NOD1, NOD2, and IFN-γ receptors. In conclusion, the low secretion of IL-8 through stimulation with agonists of TLR2, TLR4, TLR7/8, TLR9, and NOD2 at t1 suggests their possible contribution to immunosuppression following hyperinflammation in COVID-19 disease.

High glucose concentrations induce TNF-α production through the down-regulation of CD33 in primary human monocytes.

BACKGROUND: CD33 is a membrane receptor containing a lectin domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that is able to inhibit cytokine production. CD33 is expressed by monocytes, and reduced expression of CD33 correlates with augmented production of inflammatory cytokines, such as IL-1ß, TNF-α, and IL-8. However, the role of CD33 in the inflammation associated with hyperglycemia and diabetes is unknown. Therefore, we studied CD33 expression and inflammatory cytokine secretion in freshly isolated monocytes from patients with type 2 diabetes. To evaluate the effects of hyperglycemia, monocytes from healthy donors were cultured with different glucose concentrations (15-50 mmol/l D-glucose), and CD33 expression and inflammatory cytokine production were assessed. The expression of suppressor of cytokine signaling protein-3 (SOCS-3) and the generation of reactive oxygen species (ROS) were also evaluated to address the cellular mechanisms involved in the down-regulation of CD33. RESULTS: CD33 expression was significantly decreased in monocytes from patients with type 2 diabetes, and higher levels of TNF-α, IL-8 and IL-12p70 were detected in the plasma of patients compared to healthy donors. Under high glucose conditions, CD33 protein and mRNA expression was significantly decreased, whereas spontaneous TNF-α secretion and SOCS-3 mRNA expression were increased in monocytes from healthy donors. Furthermore, the down-regulation of CD33 and increase in TNF-α production were prevented when monocytes were treated with the antioxidant α-tocopherol and cultured under high glucose conditions. CONCLUSION: Our results suggest that hyperglycemia down-regulates CD33 expression and triggers the spontaneous secretion of TNF-α by peripheral monocytes. This phenomenon involves the generation of ROS and the up-regulation of SOCS-3. These observations support the importance of blood glucose control for maintaining innate immune function and suggest the participation of CD33 in the inflammatory profile associated with type 2 diabetes.

CD33 is downregulated by influenza virus H1N1pdm09 and induces ROS and the TNF-α, IL-1ß, and IL-6 cytokines in human mononuclear cells.

The influenza A virus (IAV) H1N1pdm09 induces exacerbated inflammation, contributing to disease complications. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), favor an inflammatory response that aids viral replication and survival. A pathway by which spontaneous TNF-α production occurs involves either the reduction of Siglec-3 (CD33) levels or the absence of its ligand, sialic acid. Influenza virus uses sialic acid to enter cells by reducing their expression; however, the role of CD33 in IAV H1N1pdm09 stimulation and its relationship with inflammation have not yet been studied. To evaluate the role of CD33 in proinflammatory cytokine production in IAV H1N1pdm09 stimulation, peripheral blood mononuclear cells from healthy subjects were incubated with IAV H1N1pdm09. We observed that the infection caused an increase in the mRNA expression of proinflammatory cytokines such as TNF-α, interleukin (IL)-1ß, and IL-6 and a significant reduction in CD33 expression by monocytes at an early stage of infection. Additionally, suppressor of cytokine signaling 3 (SOCS-3) mRNA expression was upregulated at 6 h, and reactive oxygen species (ROS) production increased at 1.5 h. Moreover, a significant reduction in CD33 expression on the cell surface of monocytes from influenza patients or of IAV H1N1pdm09-stimulated monocytes incubated in vitro was observed by flow cytometry. The results suggest that the decrease in CD33 and increase of SOCS-3 expression induced by IAV H1N1pdm09 triggered TNF-α secretion and ROS production, suggesting an additional way to exacerbate inflammation during viral infection.

Mycobacterium tuberculosis whiB3 and Lipid Metabolism Genes Are Regulated by Host Induced Oxidative Stress

The physiological state of the human macrophage may impact the metabolism and the persistence of Mycobacterium tuberculosis. This pathogen senses and counters the levels of O2, CO, reactive oxygen species (ROS), and pH in macrophages. M. tuberculosis responds to oxidative stress through WhiB3. The goal was to determine the effect of NADPH oxidase (NOX) modulation and oxidative agents on the expression of whiB3 and genes involved in lipid metabolism (lip-Y, Icl-1, and tgs-1) in intracellular mycobacteria. Human macrophages were first treated with NOX modulators such as DPI (ROS inhibitor) and PMA (ROS activator), or with oxidative agents (H2O2 and generator system O2•-), and then infected with mycobacteria. We determined ROS production, cell viability, and expression of whiB3, as well as genes involved in lipid metabolism. PMA, H2O2, and O2•- increased ROS production in human macrophages, generating oxidative stress in bacteria and augmented the gene expression of whiB3, lip-Y, Icl-1, and tgs-1. Our results suggest that ROS production in macrophages induces oxidative stress in intracellular bacteria inducing whiB3 expression. This factor may activate the synthesis of reserve lipids produced to survive in the latency state, which allows its persistence for long periods within the host.

Sex-Dependent Differential Expression of Lipidic Mediators Associated with Inflammation Resolution in Patients with Pulmonary Tuberculosis.

There is a sex bias in tuberculosis's severity, prevalence, and pathogenesis, and the rates are higher in men. Immunological and physiological factors are fundamental contributors to the development of the disease, and sex-related factors could play an essential role in making women more resistant to severe forms of the disease. In this study, we evaluated sex-dependent differences in inflammatory markers. Serum samples were collected from 34 patients diagnosed with pulmonary TB (19 male and 15 female) and 27 healthy controls (18 male and 9 female). Cytokines IL2, IL4, IL6, IL8, IL10, IFNγ, TNFα, and GM-CSF, and eicosanoids PGE2, LTB4, RvD1, and Mar1 were measured using commercially available immunoassays. The MDA, a product of lipidic peroxidation, was measured by detecting thiobarbituric-acid-reactive substances (TBARS). Differential inflammation patterns between men and women were observed. Men had higher levels of IL6, IL8, and TNFα than women. PGE2 and LTB4 levels were higher in patients than healthy controls, but there were no differences for RvD1 and Mar1. Women had higher RvD1/PGE2 and RvD1/LTB4 ratios among patients. RvD1 plays a vital role in resolving the inflammatory process of TB in women. Men are the major contributors to the typical pro-inflammatory profile observed in the serum of tuberculosis patients.

High Vitamin D Concentrations Restore the Ability to Express LL37 by M. tuberculosis-Infected Human Macrophages.

Vitamin D has an immunomodulatory function and is involved in eliminating pathogens. Vitamin D deficiencies reported in Type 2 diabetes mellitus (T2DM) patients make them more susceptible to developing tuberculosis (TB). The macrophages are the immune cells that control intracellular pathogens by producing the antimicrobial peptide cathelicidin-LL37. This pathway involves TLR activation by pathogens, vitamin D receptor (VDR) ligation, and the enzyme 1α-hydroxylase Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1). However, it is not clear whether the biological actions of vitamin D are affected by high glucose concentrations. This study aimed to evaluate the vitamin D contribution in the expression of VDR and CYP27B1, involved in the conversion of an inactive to an active form of vitamin D in the infected macrophages using M. tuberculosis as an infection model. The expression of LL37 and the nucleus translocation of VDR were evaluated as the readout of the response of vitamin D and determined if those processes are affected by glucose concentrations. Macrophages from healthy donors were cultured under glucose concentrations of 5.5, 15, or 30 mM, stimulated with vitamin D in inactive (25(OH)D3) or active (1,25(OH)2D3) forms, and infected with M. tuberculosis. The vitamin D-dependent induction of LL37 and the expression of VDR and CYP27B1 genes were analyzed by qPCR, and VDR translocation was analyzed in nuclear protein extracts by ELISA. M. tuberculosis downregulated the expression of LL37 regardless of the glucose concentration, whereas VDR and CYP27B1 upregulated it regardless of the glucose concentration. After evaluating two concentrations of vitamin D, 1 nM or 1 µM, the high concentration (1 µM) was necessary to restore the induction of LL37 expression in M. tuberculosis-infected macrophages. High concentrations of the inactive form of vitamin D restore the infected macrophages' ability to express LL37 regardless of the glucose concentration. This finding supports the idea that vitamin D administration in patients with T2DM could benefit TB control and prevention.

Human Pulmonary Tuberculosis: Understanding the Immune Response in the Bronchoalveolar System.

Mycobacterium tuberculosis, the causal agent of one of the most devastating infectious diseases worldwide, can evade or modulate the host immune response and remain dormant for many years. In this review, we focus on identifying the local immune response induced in vivo by M. tuberculosis in the lungs of patients with active tuberculosis by analyzing data from untouched cells from bronchoalveolar lavage fluid (BALF) or exhaled breath condensate (EBC) samples. The most abundant resident cells in patients with active tuberculosis are macrophages and lymphocytes, which facilitate the recruitment of neutrophils. The cellular response is characterized by an inflammatory state and oxidative stress produced mainly by macrophages and T lymphocytes. In the alveolar microenvironment, the levels of cytokines such as interleukins (IL), chemokines, and matrix metalloproteinases (MMP) are increased compared with healthy patients. The production of cytokines such as interferon (IFN)-γ and IL-17 and specific immunoglobulin (Ig) A and G against M. tuberculosis indicate that the adaptive immune response is induced despite the presence of a chronic infection. The role of epithelial cells, the processing and presentation of antigens by macrophages and dendritic cells, as well as the role of tissue-resident memory T cells (Trm) for in situ vaccination remains to be understood.

High Glucose Concentrations Impair the Processing and Presentation of Mycobacterium tuberculosis Antigens In Vitro.

Type 2 diabetes is an established risk factor for tuberculosis, but the underlying mechanisms are largely unknown. We established an in vitro model to analyze the effect of high glucose concentrations in antigen processing and presentation in antigen-presenting cells. Human monocyte-derived macrophages (MDMs) were exposed to high (11 mM and 30 mM) and low (5.5 mM) glucose concentrations and infected with Mycobacterium tuberculosis (Mtb). Flow cytometry was used to analyze the effect of high glucose concentrations in histocompatibility complex (MHC) class II molecules (HLA-DR) and co-stimulatory molecules (CD80 and CD86), indispensable for an adequate antigenic presentation and CD4+ T cell activation. HLA-DR and CD86 were significantly decreased by high glucose concentrations compared with low glucose concentrations. Confocal microscopy was used to detect Rab 5 and Lamp-1, proteins involved in the kinetics of antigen processing as early markers, and Rab 7 and cathepsin D as late markers. We observed a delay in the dynamics of the acquisition of Rab 7 and cathepsin D in high glucose concentrations. Moreover, the kinetics of the formation M. tuberculosis peptide-MHC II complexes in MDMs was decreased under high glucose concentrations, reducing their capacity for T cell activation. These findings suggest that high glucose concentrations directly affect antigenic processing, and therefore antigenic presentation.

Oxidative Stress and Inflammatory Mediators in Exhaled Breath Condensate of Patients with Pulmonary Tuberculosis. A Pilot Study with a Biomarker Perspective.

Tuberculosis (TB) is one of the highest infectious burdens worldwide. An excess of inflammation and inadequate antioxidant defense mechanisms are believed to lead to chronic inflammation and lung damage in tuberculosis (TB). However, circulating metabolites do not always replicate lung-associated biomarkers that define the pathobiology of the disease. The objective of this study was to determine the utility of exhaled breath condensate (EBC), a non-invasive and straightforward sample, to evaluate alveolar space-derived metabolites of redox state and inflammation. We assessed the levels of exhaled oxidant/antioxidant parameters (8-isoprostane, MDA, GSH), inflammatory markers, such as nucleosomes, cytokines (IL-2, IL-4, IL-6 and IL-8, IL-10, GM-CSF, TNF-α, and IFN-γ) and lipid mediators (PGE2, LTB4, RvD1, and Mar1), in patients with recently diagnosed pulmonary TB and healthy controls' EBC and serum. The TB patients showed 36% lower GSH levels, and 2-, 1.4-, 1.1-, and 50-fold higher levels of 8-isoprostanes, nucleosomes, IL-6, and LTB4, respectively, in EBC. There was no correlation between EBC and serum, highlighting the importance of measuring local biomarkers. Quantitation of local inflammatory molecules and redox states in EBC would help find biomarkers useful for pharmacological and follow-up studies in pulmonary tuberculosis.

CXCL17 Is Dispensable during Hypervirulent Mycobacterium tuberculosis HN878 Infection in Mice.

CXCL17 is a novel mucosal chemokine that mediates myeloid cell recruitment and bactericidal activity and highly expressed in the respiratory tract. However, its role in tuberculosis (TB) immunopathogenesis or protection remains unknown. In this study, we evaluated the function of CXCL17 in a mouse model of aerosol infection with the clinical W-Beijing lineage Mycobacterium tuberculosis hypervirulent HN878 strain. Our results show that CXCL17 production increases in the lung of M. tuberculosis-infected mice during acute and chronic stages of infection. Moreover, in vitro M. tuberculosis infection of epithelial cells and myeloid cells induces production of CXCL17. In humans, lower serum CXCL17 levels are observed among active pulmonary TB patients when compared with subjects with latent TB infection and healthy controls, suggesting a protective role. However, mice treated with rCXCL17 show similar lung bacterial burden and inflammation compared with control animals, despite an increased lung myeloid cell accumulation. Finally, CXCL17-/- mice are not more susceptible to TB than wild-type animals. These findings suggest that CXCL17 is induced in both murine epithelial and myeloid cells upon M. tuberculosis infection and increased expression during human latent TB infection. However, CXCL17 may have a dispensable role during pulmonary TB.

A Dual Marker for Monitoring MDR-TB Treatment: Host-Derived miRNAs and M. tuberculosis-Derived RNA Sequences in Serum.

Background: In the absence of a late marker of treatment failure or relapse in MDR-TB patients, biomarkers based on host-miRNAs coupled with M. tuberculosis-RNAs evaluated in extracellular vesicles (EVs) are an alternative follow-up for MDR-TB disease. Characterization of EVs cargo to identify differentially expressed miRNAs before and after treatment, and to identify M. tuberculosis-derived RNA in serum EVs from resistant TB patients. Methods: EVs were isolated from serum of 26 drug-resistant TB (DR-TB) patients and 16 healthy subjects. Differential expression of miRNAs in pooled exosomes from both untreated and treated patients was assessed and individually validated at different time points during treatment. In addition, M. tuberculosis RNA was amplified in the same samples by qPCR. Results: A multivariate analysis using miR-let-7e-5p, -197-3p and -223-3p were found to be a more sensitive discriminator between healthy individuals and those with TB for both DR-TB (AUC= 0.96, 95%, CI=0.907-1) and MDR-TB groups (AUC= 0.95, 95%, CI= 0.89-1). Upregulation of miR-let-7e-5p were observed at the time of M. tuberculosis negative culture T(3-5) for MDR-TB group or for long-term T(9-15) for MDR-TB group without diabetes (T2DM). A second pathogen-based marker based on 30kDa and 5KST sequences was detected in 33% of the MDR-TB patients after the intensive phase of treatment. The miR-let7e-5p is a candidate biomarker for long-term monitoring of treatment for the group of MDR-TB without T2DM. A dual marker of host-derived miR-let7e-5p and M. tuberculosis-derived RNA for monitoring-TB treatment based in serum EVs. Conclusion: A dual marker consisting of host-derived miR-let7e-5p and M. tuberculosis-derived RNA, could be an indicator of treatment failure or relapse time after treatment was completed.

Human serum proteins bind to Sporothrix schenckii conidia with differential effects on phagocytosis.

Serum is an important source of proteins that interact with pathogens. Once bound to the cell surface, serum proteins can stimulate the innate immune system. The phagocytosis of Sporothrix schenckii conidia by human macrophages is activated through human serum opsonisation. In this study, we have attempted to characterise human blood serum proteins that bind to the cell wall of S. schenckii conidia. We systematically observed the same four proteins independent of the plasma donor: albumin, serum amyloid protein (SAP), α-1 antitrypsin (AAT), and transferrin were identified with the help of tandem mass spectrometry. Phagocytosis depended on the concentration of the SAP or α-1 antitrypsin that was used to opsonise the conidia; however, transferrin or albumin did not have any effect on conidia internalisation. The presence of mannose did not affect macrophage phagocytosis of the conidia opsonised with SAP or α-1 antitrypsin, which suggests that these proteins are not recognised by the mannose receptor.