IL-10 predicts incident neuroinflammatory disease and proviral load dynamics in a large Brazilian cohort of people living with human T-lymphotropic virus type 1.

Human T-Lymphotropic Virus type-1 (HTLV-1) is a unique retrovirus associated with both leukemogenesis and a specific neuroinflammatory condition known as HTLV-1-Associated Myelopathy (HAM). Currently, most proposed HAM biomarkers require invasive CSF sampling, which is not suitable for large cohorts or repeated prospective screening. To identify non-invasive biomarkers for incident HAM in a large Brazilian cohort of PLwHTLV-1 (n=615 with 6,673 person-years of clinical follow-up), we selected all plasma samples available at the time of entry in the cohort (between 1997-2019), in which up to 43 cytokines/chemokines and immune mediators were measured. Thus, we selected 110 People Living with HTLV-1 (PLwHTLV-1), of which 68 were neurologically asymptomatic (AS) at baseline and 42 HAM patients. Nine incident HAM cases were identified among 68 AS during follow-up. Using multivariate logistic regression, we found that lower IL-10, IL-4 and female sex were independent predictors of clinical progression to definite HAM (AUROC 0.91), and outperformed previously suggested biomarkers age, sex and proviral load (AUROC 0.77). Moreover, baseline IL-10 significantly predicted proviral load dynamics at follow-up in all PLwHTLV-1. In an exploratory analysis, we identified additional plasma biomarkers which were able to discriminate iHAM from either AS (IL6Rα, IL-27) or HAM (IL-29/IFN-λ1, Osteopontin, and TNFR2). In conclusion, female sex and low anti-inflammatory IL-10 and IL-4 are independent risk factors for incident HAM in PLwHTLV-1,while proviral load is not, in agreement with IL-10 being upstream of proviral load dynamics. Additional candidate biomarkers IL-29/IL-6R/TNFR2 represent plausible therapeutic targets for future clinical trials in HAM patients.

An integrated analysis of the structural changes and gene expression of spleen in human visceral leishmaniasis with and without HIV coinfection.

The spleen plays a pivotal role in the pathogenesis of visceral leishmaniasis. In severe forms of the disease, the spleen undergoes changes that can compromise its function in surveilling blood-circulating pathogens. In this study, we present an integrated analysis of the structural and gene expression alterations in the spleens of three patients with relapsing visceral leishmaniasis, two of whom were coinfected with HIV. Our findings reveal that the IL6 signaling pathway plays a significant role in the disorganization of the white pulp, while BCL10 and ICOSLG are associated with spleen organization. Patients coinfected with HIV and visceral leishmaniasis exhibited lower splenic CD4+ cell density and reduced expression of genes such as IL15. These effects may contribute to a compromised immune response against L. infantum in coinfected individuals, further impacting the structural organization of the spleen.

High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil.

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.

Genomic Detection of the Emerging, Highly Pathogenic HIV-1 Subtype D in Bahia, Northeast Brazil.

(1) Background: The HIV subtype D is generally associated with a faster decline in CD4+ T cell counts, a higher viral load, and a faster progression to AIDS. However, it is still poorly characterized in Brazil. In this study, we used genomics and epidemiological data to investigate the transmission dynamics of HIV subtype D in the state of Bahia, Northeast Brazil. (2) Methods: To achieve this goal, we obtained four novel HIV-1 subtype D partial pol genome sequences using the Sanger method. To understand the emergence of this novel subtype in the state of Bahia, we used phylodynamic analysis on a dataset comprising 3704 pol genome sequences downloaded from the Los Alamos database. (3) Results: Our analysis revealed three branching patterns, indicating multiple introductions of the HIV-1 subtype D in Brazil from the late 1980s to the late 2000s and a single introduction event in the state of Bahia. Our data further suggest that these introductions most likely originated from European, Eastern African, Western African, and Southern African countries. (4) Conclusion: Understanding the distribution of HIV-1 viral strains and their temporal dynamics is crucial for monitoring the real-time evolution of circulating subtypes and recombinant forms, as well as for designing novel diagnostic and vaccination strategies. We advocate for a shift to active surveillance, to ensure adequate preparedness for future epidemics mediated by emerging viral strains.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Systemic cytokines and GlycA discriminate disease status and predict corticosteroid response in HTLV-1-associated neuroinflammation.

BACKGROUND: HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is an incapacitating neuroinflammatory disorder for which no disease-modifying therapy is available, but corticosteroids provide some clinical benefit. Although HAM/TSP pathogenesis is not fully elucidated, older age, female sex and higher proviral load are established risk factors. We investigated systemic cytokines and a novel chronic inflammatory marker, GlycA, as possible biomarkers of immunopathogenesis and therapeutic response in HAM/TSP, and examined their interaction with established risk factors. PATIENTS AND METHODS: We recruited 110 People living with HTLV-1 (PLHTLV-1, 67 asymptomatic individuals and 43 HAM/TSP patients) with a total of 946 person-years of clinical follow-up. Plasma cytokine levels (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF) and GlycA were quantified by Cytometric Bead Array and 1NMR, respectively. Cytokine signaling and prednisolone response were validated in an independent cohort by nCounter digital transcriptomics. We used multivariable regression, machine learning algorithms and Bayesian network learning for biomarker identification. RESULTS: We found that systemic IL-6 was positively correlated with both age (r = 0.50, p < 0.001) and GlycA (r = 0.45, p = 0.00049) in asymptomatics, revealing an 'inflammaging" signature which was absent in HAM/TSP. GlycA levels were higher in women (p = 0.0069), but cytokine levels did not differ between the sexes. IFN-γ (p = 0.007) and IL-17A (p = 0.0001) levels were increased in untreated HAM/TSP Multivariable logistic regression identified IL-17A and proviral load as independent determinants of clinical status, resulting in modest accuracy of predicting HAM/TSP status (64.1%), while a machine learning-derived decision tree classified HAM/TSP patients with 90.7% accuracy. Pre-treatment GlycA and TNF levels significantly predicted clinical worsening (measured by Osame Motor Disability Scale), independent of proviral load. In addition, a poor prednisolone response was significantly correlated with higher post-treatment IFN-γ levels. Likewise, a transcriptomic IFN signaling score, significantly correlated with previously proposed HAM/TSP biomarkers (CASP5/CXCL10/FCGR1A/STAT1), was efficiently blunted by in vitro prednisolone treatment of PBMC from PLHTLV-1 and incident HAM/TSP. CONCLUSIONS: An age-related increase in systemic IL-6/GlycA levels reveals inflammaging in PLHTLV-1, in the absence of neurological disease. IFN-γ and IL-17A are biomarkers of untreated HAM/TSP, while pre-treatment GlycA and TNF predict therapeutic response to prednisolone pulse therapy, paving the way for a precision medicine approach in HAM/TSP.

Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

Differential expression of polyamine biosynthetic pathways in skin lesions and in plasma reveals distinct profiles in diffuse cutaneous leishmaniasis.

Tegumentary leishmaniasis (TL) is a parasitic disease that can result in wide spectrum clinical manifestations. It is necessary to understand host and parasite determinants of clinical outcomes to identify novel therapeutic targets. Previous studies have indicated that the polyamine biosynthetic pathway is critical for Leishmania growth and survival. Despite its importance, expression of the such pathway has not been previously investigated in TL patients. We performed an exploratory analysis employing Systems Biology tools to compare circulating polyamines and amino acid concentration as well as polyamine pathway gene expression in cutaneous lesions patients presenting with distinct TL disease presentations. Diffuse cutaneous leishmaniasis (DCL) was associated with higher concentrations of amino acids, polyamines and its substrate transporters than mucosal cutaneous leishmaniasis or localized cutaneous leishmaniasis. In addition, the RNA expression of polyamine-related genes of patients lesions from two separate cohorts demonstrated that differential activation of this pathway is associated with parasite loads and able to discriminate the clinical spectrum of TL. Taken together, our findings highlight a new aspect of DCL immunopathogenesis indicating that the polyamine pathway may be explored as a novel therapeutic target to control disease burden.

Effects of 10-valent pneumococcal conjugate (PCV10) vaccination on the nasopharyngeal microbiome.

Pathogenic bacteria, such as Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis, are important vaccine targets. The 10-valent pneumococcal conjugate vaccine (PCV10) acts on 10 differents S. pneumoniae serovars. However, this vaccine could also act on other bacteria genera, leading to dysbiosis. Moreover, the vaccination has also been associated with imbalances in the ratio between commensal and potentially pathogenic bacteria. Despite the wealth of studies assessing the influence of the microbiome on vaccine effects, how vaccination can influence the microbiome remains poorly understood. Herein, we assessed the effects of PCV10 on infant nasopharyngeal microbiome composition. Nasopharyngeal aspirates were collected from children with acute respiratory infection (ARI) aged 6-23 months. Two groups were composed of 48 vaccinated and 36 unvaccinated subjects. 16S ribosomal RNA sequencing was performed to assess bacterial composition and results were analyzed with QIIME. Similar bacterial compositions were observed in the unvaccinated and vaccinated samples. Principal component analysis also indicated a similar bacterial composition between the groups. In addition, bacterial diversity was not different between the vaccinated and unvaccinated samples. Accordingly, our results suggest that PCV10 vaccination promotes a specific response against its targets, thereby preserving the nosocomial microbiome. Although not statistically significant, Streptococcus and Haemophilus genera were increased in the vaccinated group, while Moraxella was decreased. Increases in Streptococcus may be associated with vaccine-target taxa replacement by non-pathogenic species. In sum, we observed that PCV10 vaccination acts by promoting a target-specific action against pathogenic bacteria and also induces commensal bacteria colonization without substantially changing the nasopharyngeal microbiome.

An evolutionary recent IFN/IL-6/CEBP axis is linked to monocyte expansion and tuberculosis severity in humans.

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.

Dichotomy in Fatal Outcomes in a Large Cohort of People Living with HTLV-1 in São Paulo, Brazil.

Background: Despite its relatively low incidence of associated diseases, Human T-cell Leukemia Virus-1 (HTLV-1) infection was reported to carry a significant risk of mortality in several endemic areas. HTLV-1-associated diseases, adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP), as well as frequent coinfections with human immunodeficiency virus (HIV), hepatitis C virus (HCV), and Strongyloides stercoralis were associated to increased morbidity and mortality of HTLV-1 infection. Objective: To determine the mortality rate and its associated variables from an open cohort started in July 1997 at the HTLV Clinic, Emilio Ribas Institute (IIER), a major infectious disease hospital in São Paulo, Brazil. Methods: Since inception up to September 2018, we admitted 727 HTLV-1-infected individuals, with a rate of 30-50 new admissions per year. All patient data, including clinical and laboratory data, were regularly updated throughout the 21-year period, using a dedicated REDCap database. The Ethical Board of IIER approved the protocol. Results: During 21 years of clinical care to people living with HTLV-1 in the São Paulo region, we recruited 479 asymptomatic HTLV-1-infected individuals and 248 HAM/TSP patients, of which 632 remained under active follow-up. During a total of 3800 person-years of follow-up (maximum follow-up 21.5 years, mean follow-up 6.0 years), 27 individuals died (median age of 51.5 years), of which 12 were asymptomatic, one ATLL patient and 14 HAM/TSP patients. HAM/TSP diagnosis (but neither age nor gender) was a significant predictor of increased mortality by univariate and multivariate (hazard ratio (HR) 5.03, 95% CI [1.96-12.91], p = 0.001) Cox regression models. Coinfection with HIV/HCV was an independent predictor of increased mortality (HR 15.08; 95% CI [5.50-41.32]; p < 0.001), with AIDS-related infections as a more frequent cause of death in asymptomatics (6/13; p = 0.033). HIV/HCV-negative fatal HAM/TSP cases were all female, with urinary tract infection and decubitus ulcer-associated sepsis as the main cause of death (8/14, p = 0.002). Conclusions: All-cause mortality among people living with HTLV-1 in São Paulo differs between asymptomatic (2.9%) and HAM/TSP patients (7.3%), independent of age and gender. We observe a dichotomy in fatal cases, with HAM/TSP and HIV/HCV coinfection as independent risk factors for death. Our findings reveal an urgent need for public health actions, as the major causes of death, infections secondary to decubitus ulcers, and immune deficiency syndrome (AIDS)-related infections, can be targeted by preventive measures.

In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection.

Acute respiratory infection (ARI) is the most frequent cause for hospitalization in infants and young children. Using multiplexed nCounter technology to digitally quantify 600 human mRNAs in parallel with 14 virus- and 5 bacterium-specific RNAs, we characterized viral and bacterial presence in nasopharyngeal aspirates (NPA) of 58 children with ARI and determined the corresponding in situ immune profiles. NPA contained different groups of organisms and these were classified into bacterial (n = 27), viral (n = 5), codetection [containing both viral and bacterial transcripts (n = 21), or indeterminate intermediate where microbial load is below threshold (n = 5)]. We then identified differentially expressed immune transcripts (DEITs) comparing NPAs from symptomatic children vs. healthy controls, and comparing children presenting NPAs with detectable microbial load vs. indeterminate. We observed a strong innate immune response in NPAs, due to the presence of evolutionarily conserved type I Interferon (IFN)-stimulated genes (ISG), which was correlated with total bacterial and/or viral load. In comparison with indeterminate NPAs, adaptive immunity transcripts discriminated among viral, bacterial, and codetected microbial profiles. In viral NPAs, B cell transcripts were significantly enriched among DEITs, while only type III IFN was correlated with viral load. In bacterial NPAs, myeloid cells and coinhibitory transcripts were enriched and significantly correlated with bacterial load. In conclusion, digital nCounter transcriptomics provide a microbial and immunological in situ "snapshot" of the nasopharyngeal interface in children with ARI. This enabled discrimination among viral, bacterial, codetection, and indeterminate transcripts in the samples using non-invasive sampling.

Leishmania braziliensis Subverts Necroptosis by Modulating RIPK3 Expression.

Leishmania braziliensis infection causes skin ulcers, typically found in localized cutaneous leishmaniasis (LCL). This tissue pathology associates with different modalities of cell necrosis, which are subverted by the parasite as a survival strategy. Herein we examined the participation of necroptosis, a specific form of programmed necrosis, in LCL lesions and found reduced RIPK3 and PGAM5 gene expression compared to normal skin. Assays using infected macrophages demonstrated that the parasite deactivates both RIPK3 and MLKL expression and that these molecules are important to control the intracellular L. braziliensis replication. Thus, LCL-related necroptosis may be targeted to control infection and disease immunopathology.

Diagnostic accuracy of digital RNA quantification versus real-time PCR for the detection of respiratory syncytial virus in nasopharyngeal aspirates from children with acute respiratory infection.

BACKGROUND: Virus-specific molecular assays such as real-time polymerase chain reaction (RT-PCR) are regularly used as the gold standard to diagnose viral respiratory tract infections, but simultaneous detection of multiple different pathogens is often challenging. A multiplex digital method of RNA quantification, nCounter (NanoString Technologies), can overcome this disadvantage and identify, in a single reaction, the presence of different respiratory viruses. OBJECTIVES: To evaluate the accuracy of nCounter to identify and quantify RSV-A and RSV-B in nasopharyngeal aspirates (NPA) of children (6-23-months-old) with acute respiratory infection. STUDY DESIGN: NPA was collected at enrolment in a prospective cross-sectional study conducted in Salvador, Brazil. A quantitative RT-PCR with a subgroup-specific primer and probeset for RSV-A and RSV-B was performed in parallel with a customized nCounter probeset containing viral targets in NPA. RESULTS: Of 559 NPA tested, RSV was detected by RT-PCR in 139 (24.9%), by nCounter in 122 (21.8%) and by any method in 158 (28.3%) cases. Compared to the gold standard of qRT-PCR, sensitivity of nCounter was 74.3% (95%CI:63.3%-82.9% RSV-A) and 77.6% (95%CI:66.3%-85.9% RSV-B); specificity was 98.4% (95%CI:96.8%-99.2% RSV-A) and 97.8% (95%CI:96.0%-98.8% RSV-B); positive predictive value was 87.3% (95%CI:76.9%-93.4% RSV-A) and 82.5% (95%CI:71.4%-90.0% RSV-B) and negative predictive value was 96.1% (95%CI:94.1%-97.5% RSV-A), and 96.9% (95%CI:95.1%-98.2% RSV-B). Accuracy was 95.2% (95%CI:93.1%-96.7%) for RSV-A and 95.3% (95%CI:93.3%-96.9%) for RSV-B, while both methods significantly correlated for RSV-A (r = 0.44, p = 8 × 10-5) and RSV-B (r = 0.73, p = 3 × 10-12) quantification. CONCLUSIONS: nCounter is highly accurate in detecting RSV-A/B in NPA. Robustness and high-throughput multiplexing indicate its use in large-scale epidemiological studies.

Comprehensive Antiretroviral Restriction Factor Profiling Reveals the Evolutionary Imprint of the ex Vivo and in Vivo IFN-ß Response in HTLV-1-Associated Neuroinflammation.

HTLV-1-Associated Myelopathy (HAM/TSP) is a progressive neuroinflammatory disorder for which no disease-modifying treatment exists. Modest clinical benefit from type I interferons (IFN-α/ß) in HAM/TSP contrasts with its recently identified IFN-inducible gene signature. In addition, IFN-α treatment in vivo decreases proviral load and immune activation in HAM/TSP, whereas IFN-ß therapy decreases tax mRNA and lymphoproliferation. We hypothesize this "IFN paradox" in HAM/TSP might be explained by both cell type- and gene-specific effects of type I IFN in HTLV-1-associated pathogenesis. Therefore, we analyzed ex vivo transcriptomes of CD4+ T cells, PBMCs and whole blood in healthy controls, HTLV-1-infected individuals, and HAM/TSP patients. First, we used a targeted approach, simultaneously quantifying HTLV-1 mRNA (HBZ, Tax), proviral load and 42 host genes with known antiretroviral (anti-HIV) activity in purified CD4+ T cells. This revealed two major clusters ("antiviral/protective" vs. "proviral/deleterious"), as evidenced by significant negative (TRIM5/TRIM22/BST2) vs. positive correlation (ISG15/PAF1/CDKN1A) with HTLV-1 viral markers and clinical status. Surprisingly, we found a significant inversion of antiretroviral activity of host restriction factors, as evidenced by opposite correlation to in vivo HIV-1 vs. HTLV-1 RNA levels. The anti-HTLV-1 effect of antiviral cluster genes was significantly correlated to their adaptive chimp/human evolution score, for both Tax mRNA and PVL. Six genes of the proposed antiviral cluster underwent lentivirus-driven purifying selection during primate evolution (TRIM5/TRIM22/BST2/APOBEC3F-G-H), underscoring the cross-retroviral evolutionary imprint. Secondly, we examined the genome-wide type I IFN response in HAM/TSP patients, following short-term ex vivo culture of PBMCs with either IFN-α or IFN-ß. Microarray analysis evidenced 12 antiretroviral genes (including TRIM5α/TRIM22/BST2) were significantly up-regulated by IFN-ß, but not IFN-α, in HAM/TSP. This was paralleled by a significant decrease in lymphoproliferation by IFN-ß, but not IFN-α treatment. Finally, using published ex vivo whole blood transcriptomic data of independent cohorts, we validated the significant positive correlation between TRIM5, TRIM22, and BST2 in HTLV-1-infected individuals and HAM/TSP patients, which was independent of the HAM/TSP disease signature. In conclusion, our results provide ex vivo mechanistic evidence for the observed immunovirological effect of in vivo IFN-ß treatment in HAM/TSP, reconcile an apparent IFN paradox in HTLV-1 research and identify biomarkers/targets for a precision medicine approach.

ISG15-Induced IL-10 Is a Novel Anti-Inflammatory Myeloid Axis Disrupted during Active Tuberculosis.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Systems Approach Reveals Nuclear Factor Erythroid 2-Related Factor 2/Protein Kinase R Crosstalk in Human Cutaneous Leishmaniasis.

Leishmania parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive in vitro and ex vivo mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of in situ (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in Leishmania amazonensis infection in vitro. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of Sod1 Nrf2-dependent gene and reduced parasite load. L. amazonensis counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 via PKR. This Nrf2/Keap1 observation was confirmed in situ in skin biopsies from Leishmania-infected patients. Next, we explored the ex vivo transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. In silico enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our in vitro findings. Our integrated in vitro, ex vivo, and in silico approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.

DETC-based bacterial cellulose bio-curatives for topical treatment of cutaneous leishmaniasis.

The treatment of leishmaniasis still relies on drugs with potentially serious adverse effects. Herein, we tested a topical formulation of bacterial cellulose (BC) membranes containing Diethyldithiocarbamate (DETC), a superoxide dismutase 1 inhibitor. Leishmania-infected macrophages exposed to BC-DETC resulted in parasite killing, without pronounced toxic effects to host cells. This outcome was associated with lower SOD1 activity and higher production of superoxide and cytokine mediators. Topical application of BC-DETC significantly decreased lesion size, parasite load and the inflammatory response at the infection site, as well as the production of both IFN-γ and TNF. Combination of topical BC-DETC plus intraperitoneal Sbv also significantly reduced disease development and parasite load. The leishmanicidal effect of BC-DETC was extended to human macrophages infected with L. braziliensis, highlighting the feasibility of BC-DETC as a topical formulation for chemotherapy of cutaneous leishmaniasis caused by L. braziliensis.

Gene Expression Profile of High IFN-γ Producers Stimulated with Leishmania braziliensis Identifies Genes Associated with Cutaneous Leishmaniasis.

BACKGROUND: The initial response to Leishmania parasites is essential in determining disease development or resistance. In vitro, a divergent response to Leishmania, characterized by high or low IFN-γ production has been described as a potential tool to predict both vaccine response and disease susceptibility in vivo. METHODS AND FINDINGS: We identified uninfected and healthy individuals that were shown to be either high- or low IFN-γ producers (HPs and LPs, respectively) following stimulation of peripheral blood cells with Leishmania braziliensis. Following stimulation, RNA was processed for gene expression analysis using immune gene arrays. Both HPs and LPs were shown to upregulate the expression of CXCL10, IFI27, IL6 and LTA. Genes expressed in HPs only (CCL7, IL8, IFI44L and IL1B) were associated with pathways related to IL17 and TREM 1 signaling. In LPs, uniquely expressed genes (for example IL9, IFI44, IFIT1 and IL2RA) were associated with pathways related to pattern recognition receptors and interferon signaling. We then investigated whether the unique gene expression profiles described here could be recapitulated in vivo, in individuals with active Cutaneous Leishmaniasis or with subclinical infection. Indeed, using a set of six genes (TLR2, JAK2, IFI27, IFIT1, IRF1 and IL6) modulated in HPs and LPs, we could successfully discriminate these two clinical groups. Finally, we demonstrate that these six genes are significantly overexpressed in CL lesions. CONCLUSION: Upon interrogation of the peripheral response of naive individuals with diverging IFN-γ production to L. braziliensis, we identified differences in the innate response to the parasite that are recapitulated in vivo and that discriminate CL patients from individuals presenting a subclinical infection.

Respiratory syncytial virus a and b display different temporal patterns in a 4-year prospective cross-sectional study among children with acute respiratory infection in a tropical city.

Respiratory syncytial virus (RSV) is one of the most common etiological agents of childhood respiratory infections globally. Information on seasonality of different antigenic groups is scarce. We aimed to describe the frequency, seasonality, and age of children infected by RSV antigenic groups A (RSVA) and B (RSVB) among children with ARI in a 4-year period.Children (6-23 months old) with respiratory infection for ≤7 days were enrolled in a prospective cross-sectional study, from September, 2009 to October, 2013, in Salvador, in a tropical region of Brazil. Upon recruitment, demographic, clinical data, and nasopharyngeal aspirates (NPA) were collected. A multiplex quantitative real-time polymerase chain reaction (RT-PCR) with a group-specific primer and probeset for RSVA and RSVB was used. Seasonal distribution of infection by RSV different antigenic groups was evaluated by Prais-Wisten regression.Of 560 cases, the mean age was 11.4 ±â€Š4.5 months and there were 287 (51.3%) girls. Overall, RSV was detected in 139 (24.8%; 95% CI: 21.4%-28.5%) cases, RSVA in 74 (13.2%; 95% CI: 10.6%-16.2%) cases, and RSVB in 67 (12.0%; 95% CI: 9.5%-14.9%) cases. Two (0.4%; 95% CI: 0.06%-1.2%) cases had coinfection. RSVA frequency was 9.6%, 18.4%, 21.6%, and 3.1% in 2010, 2011, 2012, and 2013, respectively. RSVB frequency was 19.2%, 0.7%, 1.4%, and 35.4% in the same years. RSVA was more frequently found from August to January than February to July (18.2% vs. 6.4%, P < 0.001). RSVB was more frequently found (P < 0.001) between March and June (36.0%) than July to October (1.0%) or November to February (1.6%). RSVB infection showed seasonal distribution and positive association with humidity (P = 0.02) whereas RSVA did not. RSVA was more common among children ≥1-year-old (17.8% vs. 1.8%; P = 0.02), as opposed to RSVB (11.5% vs. 12.2%; P = 0.8).One quarter of patients had RSV infection. RSVA compromised more frequently children aged ≥1 year. RSVA predominated in 2011 and 2012 whereas RSVB predominated in 2010 and 2013. In regard to months, RSVA was more frequent from August to January whereas RSVB was more often detected between March and June. Markedly different monthly as well as yearly patterns for RSVA and RSVB reveal independent RSV antigenic groups' epidemics.