Corrigendum: Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

[This corrects the article DOI: 10.3389/fimmu.2018.02558.].

Tuberculosis-associated anemia is linked to a distinct inflammatory profile that persists after initiation of antitubercular therapy.

Pulmonary tuberculosis (PTB) is associated with chronic inflammation and anemia. How anemia impacts systemic inflammation in PTB patients undergoing antitubercular therapy (ATT) is not fully understood. In the present study, data on several blood biochemical parameters were retrospectively analyzed from 118 PTB patients during the first 60 days of ATT. Multidimensional statistical analyses were employed to perform detailed inflammatory profiling of patients stratified by anemia status prior to treatment. Anemia was defined as hemoglobin levels <12.5 g/dL for female and <13.5 g/dL for male individuals. The findings revealed that most of anemia cases were likely caused by chronic inflammation. A distinct biosignature related to anemia was detected, defined by increased values of uric acid, C-reactive protein, and erythrocyte sedimentation rate. Importantly, anemic patients sustained increased levels of several biochemical markers at day 60 of therapy. Preliminary analysis failed to demonstrate association between persistent inflammation during ATT with frequency of positive sputum cultures at day 60. Thus, TB patients with anemia exhibit a distinct inflammatory profile, which is only partially reverted at day 60 of ATT.

Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).

Emergence of Polyfunctional Cytotoxic CD4+ T Cells in Mycobacterium avium Immune Reconstitution Inflammatory Syndrome in Human Immunodeficiency Virus-Infected Patients.

Background: Immune reconstitution inflammatory syndrome (IRIS) is an aberrant inflammatory response in individuals with advanced human immunodeficiency virus (HIV) infection, after antiretroviral therapy (ART) initiation. The pathogenesis of Mycobacterium avium complex (MAC)-associated IRIS has not been fully elucidated. Methods: We investigated monocyte and CD4+ T-cell responses in vitro, tumor necrosis factor (TNF) expression in tissues, and plasma cytokines and inflammatory markers, in 13 HIV-infected patients with MAC-IRIS and 14 HIV-uninfected patients with pulmonary MAC infection. Results: Prior to ART, HIV-infected compared with HIV-uninfected patients, had reduced TNF+ monocytes (P = .013), although similar cytokine (interferon gamma [IFN-γ], TNF, interleukin 2 [IL-2], and interleukin 17 [IL-17])-expressing CD4+ T cells. During IRIS, monocyte cytokine production was restored. IFN-γ+ (P = .027), TNF+ (P = .004), and polyfunctional CD4+ T cells (P = 0.03) also increased. These effectors were T-betlow, and some expressed markers of degranulation and cytotoxic potential. Blockade of cytotoxic T-lymphocyte associated protein 4 and lymphocyte activation gene-3 further increased CD4+ T-cell cytokine production. Tissue immunofluorescence showed higher proportions of CD4+ and CD68+ (monocyte/macrophage) cells expressed TNF during IRIS compared with HIV-uninfected patients. Plasma IFN-γ (P = .048), C-reactive protein (P = .008), and myeloperoxidase (P < .001) levels also increased, whereas interleukin 10 decreased (P = .008) during IRIS. Conclusions: Advanced HIV infection was associated with impaired MAC responses. Restoration of monocyte responses and expansion of polyfunctional MAC-specific T-betlow CD4+ T cells with cytotoxic potential after ART initiation may overwhelm existing regulatory and inhibitory mechanisms, leading to MAC-IRIS.

Clinical and immunopathological findings during long term follow-up in Leishmania infantum experimentally infected dogs.

Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is transmitted by Lutzomyia longipalpis. While prospective clinical and immunological assessments of dogs experimentally challenged with L. infantum have been previously reported over a relatively short follow-up period, the long-term characterization of infected animals has not been performed to date. We evaluated dogs in a subclinical state for six years following experimental infection with L. infantum and Lu. longipalpis saliva, via an intradermal route, to characterize clinical, parasitological and immunological parameters arising from L. infantum experimental infection. We also assess these parameters in a group of naturally infected animals. The immune profiles of the experimentally and naturally infected animals exhibited increases of IFN-γ, IL-6 and IL-18, and decreases in TNF, IL-2, IL-8 and CXCL1, compared to controls. Our results indicate that over a six-year follow-up post-challenge, subclinically infected dogs presented low CVL clinical scores despite the persistence of Leishmania parasites in the lymph nodes, spleen and skin. Similarities observed among immune profiles in the context of experimental and natural infection seem to suggest that an enduring activation of the host immune response may lead to the control of parasite growth, thereby limiting disease severity.

Anti-parasite therapy drives changes in human visceral leishmaniasis-associated inflammatory balance.

Visceral leishmaniasis (VL) remains a major public health problem worldwide. Cytokine balance is thought to play a critical role in the development of this disease. Here, we perform a prospective exploratory study addressing whether simultaneous assessment of circulating levels of different lipid mediators and cytokines could highlight specific pathways involved with VL pathogenesis. VL patients displayed substantial increases in serum levels of Prostaglandin F2α (PGF2α), Leukotriene B4 (LTB4), Resolvin D1 (RvD1), IL-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α compared with uninfected endemic control group, while exhibiting decreased levels of TGF-ß1. Hierarchical cluster analysis of the prospective changes in the expression level of theses parameters upon anti-Leishmania treatment initiation revealed that the inflammatory profile observed in active disease gradually changed over time and was generally reversed at day 30 of therapy. Furthermore, not only the individual concentrations of most of the inflammatory biomarkers changed upon treatment, but the correlations between those and several biochemical parameters used to characterize VL disease activity were also modified over time. These results demonstrate that an inflammatory imbalance hallmarks active VL disease and open perspective for manipulation of these pathways in future studies examining a potential host-directed therapy against VL.

Systems Immunology of Diabetes-Tuberculosis Comorbidity Reveals Signatures of Disease Complications.

Comorbid diabetes mellitus (DM) increases tuberculosis (TB) risk and adverse outcomes but the pathological interactions between DM and TB remain incompletely understood. We performed an integrative analysis of whole blood gene expression and plasma analytes, comparing South Indian TB patients with and without DM to diabetic and non-diabetic controls without TB. Luminex assay of plasma cytokines and growth factors delineated a distinct biosignature in comorbid TBDM in this cohort. Transcriptional profiling revealed elements in common with published TB signatures from cohorts that excluded DM. Neutrophil count correlated with the molecular degree of perturbation, especially in TBDM patients. Body mass index and HDL cholesterol were negatively correlated with molecular degree of perturbation. Diabetic complication pathways including several pathways linked to epigenetic reprogramming were activated in TBDM above levels observed with DM alone. Our data provide a rationale for trials of host-directed therapies in TBDM, targeting neutrophilic inflammation and diabetic complication pathways to address the greater morbidity and mortality associated with this increasingly prevalent dual burden of communicable and non-communicable diseases.

Mycobacterium tuberculosis Induction of Heme Oxygenase-1 Expression Is Dependent on Oxidative Stress and Reflects Treatment Outcomes.

The antioxidant enzyme heme oxygenase-1 (HO-1) is implicated in the pathogenesis of tuberculosis (TB) and has been proposed as a biomarker of active disease. Nevertheless, the mechanisms by which Mycobacterium tuberculosis (Mtb) induces HO-1 as well as how its expression is affected by HIV-1 coinfection and successful antitubercular therapy (ATT) are poorly understood. We found that HO-1 expression is markedly increased in rabbits, mice, and non-human primates during experimental Mtb infection and gradually decreased during ATT. In addition, we examined circulating concentrations of HO-1 in a cohort of 130 HIV-1 coinfected and uninfected pulmonary TB patients undergoing ATT to investigate changes in expression of this biomarker in relation to HIV-1 status, radiological disease severity, and treatment outcome. We found that plasma levels of HO-1 were elevated in untreated HIV-1 coinfected TB patients and correlated positively with HIV-1 viral load and negatively with CD4+ T cell count. In both HIV-1 coinfected and Mtb monoinfected patients, HO-1 levels were substantially reduced during successful TB treatment but not in those who experienced treatment failure or subsequently relapsed. To further delineate the molecular mechanisms involved in induction of HO-1 by Mtb, we performed a series of in vitro experiments using mouse and human macrophages. We found that Mtb-induced HO-1 expression requires NADPH oxidase-dependent reactive oxygen species production induced by the early-secreted antigen ESAT-6, which in turn triggers nuclear translocation of the transcription factor NRF-2. These observations provide further insight into the utility of HO-1 as a biomarker of both disease and successful therapy in TB monoinfected and HIV-TB coinfected patients and reveal a previously undocumented pathway linking expression of the enzyme with oxidative stress.

Sustained elevated levels of C-reactive protein and ferritin in pulmonary tuberculosis patients remaining culture positive upon treatment initiation.

BACKGROUND: Clinical trials that evaluate new anti-tubercular drugs and treatment regimens take years to complete due to the slow clearance of Mycobacterium tuberculosis infection and the lack of early biomarkers that predict treatment outcomes. Host Inflammation markers have been associated with tuberculosis (TB) pathogenesis. In the present study, we tested if circulating levels of C-reactive protein (CRP) and ferritin reflect mycobacterial loads and inflammation in pulmonary TB (PTB) patients undergoing anti-tuberculous therapy (ATT). METHODS: Prospective measurements of CRP and ferritin, used as readouts of systemic inflammation, were performed in cryopreserved serum samples from 165 Brazilian patients with active PTB initiating ATT. Associations between levels of these laboratory parameters with mycobacterial loads in sputum as well as with sputum conversion at day 60 of ATT were tested. RESULTS: Circulating levels of both ferritin and CRP gradually decreased over time on ATT. At pre-treatment, concentrations of these parameters were unable to distinguish patients with positive from those with negative acid-fast bacilli (AFB) in sputum cultures. However, patients who remained with positive cultures at day 60 of ATT exhibited heightened levels of these inflammatory markers compared to those with negative cultures at that time point. CONCLUSIONS: CRP and Ferritin levels in serum may be useful to identify patients with positive cultures at day 60 of ATT.

Role of LTA4H Polymorphism in Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome Occurrence and Clinical Severity in Patients Infected with HIV.

BACKGROUND: Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory phenomenon complicating HIV management in coincidental tuberculosis (TB) infection, upon immune reconstitution driven by antiretroviral therapy (ART). Leukotriene A4 hydroxylase (LTA4H), an enzyme which converts LTA4 to LTB4, regulates the balance between the anti-inflammatory lipoxins and pro-inflammatory LTB4, with direct implications in TB-driven inflammation. In humans, a single nucleotide polymorphism (SNP) in the LTA4H promoter which regulates its transcriptional activity (rs17525495) has been identified and described to impact clinical severity of TB presentation and response to corticosteroid therapy. Notably, the role of LTA4H on TB-IRIS has not been previously evaluated. Here, we performed an exploratory investigation testing the association of LTA4H polymorphism with respect to frequency of TB-IRIS occurrence and severity of TB-IRIS presentation in HIV-TB co-infected individuals. METHODS: Genotypic evaluation of the LTA4H enzyme from available samples was retrospectively correlated with clinical data captured in case sheets including IRIS details. The cohort included patients recruited from a prospective cohort study nested within a randomized clinical trial (NCT0933790) of ART-naïve HIV+ patients with newly diagnosed rifampicin sensitive pulmonary TB in South India. Frequency of the wild type genotype (CC), as well as of the mutant genotypes (CT or TT) in the IRIS and non-IRIS patients was estimated. Comparative analyses were performed between wild genotype (CC) and the mutant genotypes (CT or TT) and tested for association between the LTA4H polymorphisms and IRIS incidence and clinical severity. RESULTS: A total of 142 eligible ART-naïve patients were included in the analyses. Eighty-six individuals exhibited the wild genotype (CC) while 56 had mutant genotypes (43-CT and only 13-TT). Variant allele frequency was 0.23 and 0.26 in non-IRIS group and in IRIS group, respectively. Upon ART initiation, 51 patients developed IRIS while 91 did not. IRIS incidence was 34% and 37% in the wild (CC) and mutant type (CT/TT), respectively (p = 0.858) with a higher frequency of severe IRIS presentation in the mutant genotype group compared to the wild type genotype (p = 0.0006). A logistic regression model confirmed the association between the presence of CT/TT genotypes and occurrence of severe IRIS. Corticosteroid therapy successfully resolved IRIS in all cases irrespective of the LTA4H genotype. CONCLUSION: A higher incidence of severe IRIS among patients with mutant LTA4H genotypes (CT and TT) was observed compared to the wild type, despite similar IRIS incidence and immune restoration in both groups. Steroids were effective in alleviating IRIS in all the genotypes.

Associations between systemic inflammation, mycobacterial loads in sputum and radiological improvement after treatment initiation in pulmonary TB patients from Brazil: a prospective cohort study.

BACKGROUND: Mycobacterium tuberculosis infection is known to cause inflammation and lung tissue damage in high-risk populations. Nevertheless, direct associations between mycobacterial loads, systemic inflammation and pulmonary lesions upon treatment initiation have not been fully characterized. In the present exploratory study, we prospectively depict the immune profile, microbial clearance and evolution of radiographic lesions in a pulmonary tuberculosis (PTB) patient cohort before and 60 days after anti-tuberculous treatment (ATT) initiation. METHODS: Circulating levels of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α) and C-reactive protein (CRP), as well as values of erythrocyte sedimentation rate (ESR) were measured in cryopreserved serum samples obtained from 73 PTB patients at pre-ATT and day 60 of treatment. Changes of the immune profile over time were compared with mycobacterial loads in sputum and culture conversion at day 60 of ATT. Additional analyses tested associations between improvement of chest radiographic lesions at day 60 and pre-treatment status of inflammation and mycobacterial loads. RESULTS: Within the inflammatory parameters evaluated, values of CRP, IL-2, IL-4, TNF-α and ESR significantly decreased upon treatment initiation. On the converse, IL-10 levels substantially increased at day 60 of ATT, whereas concentrations of IL-6 and IFN-γ remained unchanged. Multidimensional analyses revealed that ESR, IL-2, IL-4 and CRP were the parameters with the highest power to discriminate individuals before and after treatment initiation. We further demonstrated that higher bacterial loads in sputum at pre-ATT were associated with increased systemic inflammation and higher risk for positive M. tuberculosis sputum cultures at day 60 of treatment. Furthermore, we found that pre-ATT mycobacterial loads in sputum and systemic inflammation synergistically associated with the status of radiographic lesions during treatment (Relative risk for chest X-ray improvement: 10.0, 95 % confidence interval: 2.4-40.0, P = 0.002). CONCLUSIONS: M. tuberculosis loads in sputum are directly associated to the status of systemic inflammation and potentially impact the immune profile, culture conversion and evolution of lung lesions upon ATT initiation.

Glucose Metabolism Disorder Is Associated with Pulmonary Tuberculosis in Individuals with Respiratory Symptoms from Brazil.

BACKGROUND: Diabetes mellitus (DM) has been associated with increased risk for pulmonary tuberculosis (PTB) in endemic settings but it is unknown whether PTB risk is also increased by pre-DM. Here, we prospectively examined the association between glucose metabolism disorder (GMD) and PTB in patients with respiratory symptoms at a tuberculosis primary care reference center in Brazil. METHODS: Oral glucose tolerance test was performed and levels of fasting plasma glucose and glycohemoglobin (HbA1c) were measured in a cohort of 892 individuals presenting with respiratory symptoms of more than two weeks duration. Patients were also tested for PTB with sputum cultures. Prevalence of pre-DM and DM (based on HbA1c) was estimated and tested for association with incident PTB. Other TB risk factors including smoking history were analyzed. RESULTS: The majority of the study population (63.1%) exhibited GMD based on HbA1c ≥5.7%. Patients with GMD had higher prevalence of PTB compared to normoglycemic patients. Individuals with DM exhibited increased frequency of TB-related symptoms and detection of acid-fast bacilli in sputum smears. Among patients with previous DM diagnosis, sustained hyperglycemia (HbA1c ≥7.0%) was associated with increased TB prevalence. Smoking history alone was not significantly associated with TB in our study population but the combination of smoking and HbA1c ≥7.0% was associated with 6 times higher odds for PTB. CONCLUSIONS: Sustained hyperglycemia and pre-DM are independently associated with active PTB. This evidence raises the question whether improving glycemic control in diabetic TB patients would reduce the risk of TB transmission and simultaneously reduce the clinical burden of disease. A better understanding of mechanisms underlying these associations, especially those suggesting that pre-DM may be a factor driving susceptibility to TB is warranted.

Diabetes Is Associated with Worse Clinical Presentation in Tuberculosis Patients from Brazil: A Retrospective Cohort Study.

BACKGROUND: The rising prevalence of diabetes mellitus (DM) worldwide, especially in developing countries, and the persistence of tuberculosis (TB) as a major public health issue in these same regions, emphasize the importance of investigating this association. Here, we compared the clinical profile and disease outcomes of TB patients with or without coincident DM in a TB reference center in Brazil. METHODS: We performed a retrospective analysis of a TB patient cohort (treatment naïve) of 408 individuals recruited at a TB primary care center in Brazil between 2004 and 2010. Data on diagnosis of TB and DM were used to define the groups. The study groups were compared with regard to TB disease presentation at diagnosis as well as to clinical outcomes such as cure and mortality rates upon anti-tuberculosis therapy (ATT) initiation. A composite score utilizing clinical, radiological and microbiological parameters was used to compare TB severity between the groups. RESULTS: DM patients were older than non-diabetic TB patients. In addition, diabetic individuals more frequently presented with cough, night sweats, hemoptysis and malaise than those without DM. The overall pattern of lung lesions assessed by chest radiographic examination was similar between the groups. Compared to non-diabetic patients, those with TB-diabetes exhibited positive acid-fast bacilli in sputum samples more frequently at diagnosis and at 30 days after ATT initiation. Notably, higher values of the TB severity score were significantly associated with TB-diabetes comorbidity after adjustment for confounding factors. Moreover, during ATT, diabetic patients required more frequent transfers to TB reference hospitals for complex clinical management. Nevertheless, overall mortality and cure rates were indistinguishable between the study groups. CONCLUSIONS: These findings reinforce the idea that diabetes negatively impacts pulmonary TB severity. Our study argues for the systematic screening for DM in TB reference centers in endemic areas.