The ubiquitin ligase TRIM32 promotes the autophagic response to Mycobacterium tuberculosis infection in macrophages.

Mycobacterium tuberculosis (Mtb) is known to evade host immune responses and persist in macrophages for long periods. A mechanism that the host uses to combat Mtb is xenophagy, a selective form of autophagy that targets intracellular pathogens for degradation. Ubiquitination of Mtb or Mtb-containing compartments is a key event to recruit the autophagy machinery and mediate the bacterial delivery to the lysosome. This event relies on the coordinated and complementary activity of different ubiquitin ligases, including PARKIN, SMURF1, and TRIM16. Because each of these factors is responsible for the ubiquitination of a subset of the Mtb population, it is likely that additional ubiquitin ligases are employed by macrophages to trigger a full xenophagic response during Mtb infection. In this study, we investigated the role TRIM proteins whose expression is modulated in response to Mtb or BCG infection of primary macrophages. These TRIMs were ectopically expressed in THP1 macrophage cell line to assess their impact on Mtb replication. This screening identified TRIM32 as a novel player involved in the intracellular response to Mtb infection, which promotes autophagy-mediated Mtb degradation. The role of TRIM32 in xenophagy was further confirmed by silencing TRIM32 expression in THP1 cells, which causes increased intracellular growth of Mtb associated to impaired Mtb ubiquitination, reduced recruitment of the autophagy proteins NDP52/CALCOCO2 and BECLIN 1/BECN1 to Mtb and autophagosome formation. Overall, these findings suggest that TRIM32 plays an important role in the host response to Mtb infection through the induction of autophagy, representing a promising target for host-directed tuberculosis therapies.

Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy.

Objective: Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods: Two cohorts were evaluated: in "study population A", plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized "COVID-19 patients" and 29 "NO COVID-19 controls" all unvaccinated. In "study population B", 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results: Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in "study population A". Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in "study population B". Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion: This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis.

Introduction: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. Materials and Methods: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. Results: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. Conclusion: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.

Mycobacterium tuberculosis Immune Response in Patients With Immune-Mediated Inflammatory Disease.

Subjects with immune-mediated inflammatory diseases (IMID), such as rheumatoid arthritis (RA), have an intrinsic higher probability to develop active-tuberculosis (TB) compared to the general population. The risk ranges from 2.0 to 8.9 in RA patients not receiving therapies. According to the WHO, the RA prevalence varies between 0.3% and 1% and is more common in women and in developed countries. Therefore, the identification and treatment of TB infection (TBI) in this fragile population is important to propose the TB preventive therapy. We aimed to study the M. tuberculosis (Mtb) specific T-cell response to find immune biomarkers of Mtb burden or Mtb clearance in patients with different TB status and different risk to develop active-TB disease. We enrolled TBI subjects as example of Mtb-containment, the active-TB as example of a replicating Mtb status, and the TBI-IMID as fragile population. To study the Mtb-specific response in a condition of possible Mtb sterilization, we longitudinally enrolled TBI subjects and active-TB patients before and after TB therapy. Peripheral blood mononuclear cells were stimulated overnight with Mtb peptides contained in TB1- and TB2-tubes of the Quantiferon-Plus kit. Then, we characterized by cytometry the Mtb-specific CD4 and CD8 T cells. In TBI-IMID, the TB therapy did not affect the ability of CD4 T cells to produce interferon-γ, tumor necrosis factor-α, and interleukin-2, their functional status, and their phenotype. The TB therapy determined a contraction of the triple functional CD4 T cells of the TBI subjects and active-TB patients. The CD45RA- CD27+ T cells stood out as a main subset of the Mtb-specific response in all groups. Before the TB-preventive therapy, the TBI subjects had higher proportion of Mtb-specific CD45RA-CD27+CD4+ T cells and the active-TB subjects had higher proportion of Mtb-specific CD45RA-CD27-CD4+ T cells compared to other groups. The TBI-IMID patients showed a phenotype similar to TBI, suggesting that the type of IMID and the IMID therapy did not affect the activation status of Mtb-specific CD4 T cells. Future studies on a larger and better-stratified TBI-IMID population will help to understand the change of the Mtb-specific immune response over time and to identify possible immune biomarkers of Mtb-containment or active replication.

A whole blood test to measure SARS-CoV-2-specific response in COVID-19 patients.

OBJECTIVES: To examine whether specific T-cell-responses to SARS-CoV-2 peptides can be detected in COVID-19 using a whole-blood experimental setting, which may be further explored as a potential diagnostic tool. METHODS: We evaluated interferon (IFN)-γ levels after stimulating whole-blood with spike and remainder-antigens peptides megapools (MP) derived from SARS-CoV-2 sequences; interleukin (IL)-1ß, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, Interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1ß, Platelet-derived growth factor (PDGF), RANTES (regulated on activation, normal T cell expressed and secreted), tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) were also evaluated. RESULTS: IFN-γ-response to spike and remainder-antigens MPs was significantly increased in 35 COVID-19 patients compared with 29 'no COVID-19' individuals (medians spike-MP: 0.26 vs 0, p = 0.0002; medians remainder-antigens-MP: 0.07 vs 0.02; p = 0.02). This response was detected independently of patients' clinical parameters. IFN-γ-response to SARS-CoV-2-unrelated antigens cytomegalovirus (CMV) and Staphylococcal Enterotoxin B (SEB) was similar in COVID-19 compared with 'no COVID-19' individuals (median CMV: 3.46 vs 5.28, p = 0.16; median SEB: 12.68 vs 15.05; p = 0.1). In response to spike-MPs in COVID-19- compared with 'no COVID-19' -individuals, we found significant higher median of IL-2 (50.08 vs 0, p = 0.0018), IFN-γ (90.16 vs 0, p = 0.01), IL-4 (0.52 vs 0, p = 0.03), IL-13 (0.84 vs 0, p = 0.007) and MCP-1 (4602 vs 359.2, p = 0.05). CONCLUSIONS: Immune response to SARS-CoV-2 peptides in a whole-blood assay is associated with COVID-19 and it is characterized by both Th1 and Th2 profile. This experimental approach may be useful for developing new T-cell based diagnostic tests for disease and vaccine settings.

Latent tuberculosis infection screening in persons newly-diagnosed with HIV infection in Italy: A multicentre study promoted by the Italian Society of Infectious and Tropical Diseases.

BACKGROUND: The Italian Society of Infectious and Tropical Diseases performed a survey on the application of guidelines for the management of persons living with HIV (PLWH), to evaluate current practice and the yield of screening for latent tuberculosis infection (LTBI) in newly-diagnosed PLWH; in addition, the offer of preventive therapy to LTBI individuals and the completion rate were analysed. MATERIALS AND METHODS: Newly-diagnosed PLWH in nine centres were evaluated retrospectively (2016/2017) using binary and multinomial logistic regression to identify factors associated with LTBI diagnostic screening and QuantiFERON (QFT) results. RESULTS: Of 801 patients evaluated, 774 were studied after excluding active TB. LTBI tests were performed in 65.5%. Prescription of an LTBI test was associated with being foreign-born (odds ratio (OR) 3.19, p < 0.001), older (for 10-year increments, OR 1.22, p = 0.034), and having a CD4 count <100 cells/mm3 vs ≥500 cells/mm3 (OR 2.30, p = 0.044). LTBI was diagnosed in 6.5% of 495 patients evaluated by QFT. Positive results were associated with being foreign-born (relative risk ratio (RRR) 30.82, p < 0.001), older (for 10-year increments, RRR 1.78, p = 0.003), and having a high CD4 count (for 100 cells/mm3 increments, RRR 1.26, p < 0.003). Sixteen LTBI individuals started TB preventive therapy and eight completed it. CONCLUSIONS: LTBI screening is inconsistently performed in newly-diagnosed PLWH. Furthermore, TB preventive therapy is not offered to all LTBI individuals and compliance is poor.

Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry.

The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.

Characterization of QuantiFERON-TB-Plus results in latent tuberculosis infected patients with or without immune-mediated inflammatory diseases.

OBJECTIVES: Screening for latent tuberculosis infection (LTBI) diagnosis is mandatory in patients with immune-mediated inflammatory diseases (IMID) requiring biologics. QuantiFERON-TB-Plus (QFT-P), an LTBI diagnostic test, measures IFN-γ after M. tuberculosis-stimulation in TB1 and TB2 tubes in which a "CD4" or a "CD4 and CD8" response is respectively elicited. Aim of this study is to compare the response to QFT-P of IMID-LTBI patients candidates to a new biological therapy vs LTBI-subjects without IMID. METHODS: We prospectively enrolled 167 subjects: 61 IMID-LTBI and 106 NON-IMID-LTBI. RESULTS: All subjects were mitogen-responders. IFN-γ production was significantly lower in IMID-LTBI-patients compared to NON-IMID-LTBI-subjects. We observed discordant TB1 and TB2 results in 6.5% of IMID-LTBI-patients and in 8% of NON-IMID-LTBI-subjects. Applying a logistic regression analysis, we found that IMID-LTBI patients had a higher probability (TB1 stimulation OR 3.32; TB2 stimulation OR 4.33) to have IFNγ results ≤0.7 IU/mL compared to NON-IMID-LTBI-subjects. Interestingly, IMID-treatment did not interfere with the distribution of IFNγ-values. CONCLUSIONS: These results indicate that IMID-LTBI-patients have a low IFN-γ response to QFT-P, a high proportion of results ranging in the grey zone and a distribution of IFNγ-values independent from the IMID-treatment. These results are important for the management of LTBI screening in IMID patients.

Antitubercular and anti-inflammatory properties screening of natural products from Plectranthus species.

AIM: Confirm the use of Plectanthus spp. plants in traditional medicine, particularly as anti-inflammatory and anti-infective agents. MATERIALS & METHODS: Compounds previously isolated from Plectranthus spp. were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quantification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages with M. tuberculosis H37Rv, using CFU counts assay, at their minimum inhibitory concentration values. Results: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scavenging in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol), previously prepared (2.1 × 105 CFU/mL), with an effect similar to the antitubercular drugs ethambutol (2.0 × 105 CFU/mL) and isoniazid (1.2 × 105 CFU/mL). CONCLUSION: The present report demonstrates the relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respiratory complaints. Also, this work suggests that further studies involving other inflammatory mediators are needed to validate the anti-inflammatory use of these medicinal plants.

Clinical isolates of the modern Mycobacterium tuberculosis lineage 4 evade host defense in human macrophages through eluding IL-1ß-induced autophagy.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected over 1.7 billion people worldwide and causes 1.4 million deaths annually. Recently, genome sequence analysis has allowed the reconstruction of Mycobacterium tuberculosis complex (MTBC) evolution, with the identification of seven phylogeographic lineages: four referred to as evolutionarily "ancient", and three "modern". The MTBC strains belonging to "modern" lineages appear to show enhanced virulence that may have warranted improved transmission in humans over ancient lineages through molecular mechanisms that remain to be fully characterized. To evaluate the impact of MTBC genetic diversity on the innate immune response, we analyzed intracellular bacterial replication, inflammatory cytokine levels, and autophagy response in human primary macrophages infected with MTBC clinical isolates belonging to the ancient lineages 1 and 5, and the modern lineage 4. We show that, when compared to ancient lineage 1 and 5, MTBC strains belonging to modern lineage 4 show a higher rate of replication, associated to a significant production of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) and induction of a functional autophagy process. Interestingly, we found that the increased autophagic flux observed in macrophages infected with modern MTBC is due to an autocrine activity of the proinflammatory cytokine IL-1ß, since autophagosome maturation is blocked by an interleukin-1 receptor antagonist. Unexpectedly, IL-1ß-induced autophagy is not disadvantageous for the survival of modern Mtb strains, which reside within Rab5-positive phagosomal vesicles and avoid autophagosome engulfment. Altogether, these results suggest that autophagy triggered by inflammatory cytokines is compatible with a high rate of intracellular bacilli replication and may therefore contribute to the increased pathogenicity of the modern MTBC lineages.

Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis.

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (MtbΔ33) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the MtbΔ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, MtbΔ33 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of MtbΔ33 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb.

Use of several immunological markers to model the probability of active tuberculosis.

Blood-based biomarkers tests are attractive alternative for diagnosing tuberculosis to assays depending on mycobacteria detection. Given several immunological markers we used logistic regression to model the probability of active tuberculosis in a cohort of patients with active or latent tuberculosis, showing an increased accuracy in distinguishing active from latent tuberculosis.

Modulation of CD4 and CD8 response to QuantiFERON-TB Plus in patients with active tuberculosis and latent tuberculosis infection followed over time during treatment.

OBJECTIVE/BACKGROUND: Interferon (IFN)-γ release assays (IGRA) are designed for diagnosing tuberculosis (TB) infection. The new IGRA, QuantiFERON-TB Plus (QFT-Plus), is based on the enzyme-linked immunosorbent assay detection of IFN-γ after stimulation with Mycobacterium tuberculosis TB1 and TB2 antigens. TB1 elicits a cellular-mediated immune (CMI) response by CD4 T cells, and TB2 contains peptides recognized by both CD4 and CD8 T cells. The aim of the study is to characterize the CMI to QFT-Plus peptides in active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). METHODS: We enrolled 7 individuals with active TB and 11 individuals with LTBI at baseline and followed them during the treatment, either for active diseases or preventive therapy. Peripheral blood mononuclear cells were stimulated with QFT-Plus antigens (TB1, TB2, and mitogen). Cytokine profile (IFN-γ, tumor necrosis factor-α, interleukin-2) and phenotype (CD45RA, CD27) of CD4 and CD8 T cells were characterized by flow cytometry. RESULTS: All the individuals responded to mitogen. CD4 T-cell responses to TB1 and TB2 were similar in both individuals with active TB and those with LTBI evaluated over time. Differently, we found a higher number of TB2-associated CD8 T-cell responders in individuals with active TB than in those with LTBI. For individuals with active TB, there was no change in the specific response overtime. Differently, in individuals with LTBI, the number of CD8 responders to QFT-Plus antigens increased during preventive treatment (TB1=5/11 [45%], TB2=5/11 [45%]) compared with that at the time of enrolment (TB1=1/11 [9%], TB2=1/11 [9%]). Moreover, we analyzed the effector memory profile of T cells responding to QFT-Plus antigens. The largest component of antigen-specific CD4 T cells (65%) had a central memory (CD45RA-CD27+) phenotype at enrolment and during follow-up. In contrast, specific CD8 T cells, which were analyzed only at follow-up because they were almost absent at baseline, were characterized by a large component with naïve (CD45RA+CD27+) phenotype (40%) and a minor component with central memory (25%) features. CONCLUSION: To our knowledge, this is the first report characterizing CD4 and CD8 T-cell responses of individuals with active TB and with LTBI, followed overtime, to QFT-Plus antigens by flow cytometry. The results, although preliminary, may help in identifying better tools for monitoring therapy, especially in those with LTBI undergoing preventive treatment.

Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts.

BACKGROUND: Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E.granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4+ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4+ T-cells producing IL-2+TNF-α+Th2+ or TNF-α+Th2+ were significantly increased in the "active stages" group compared to the "inactive stages" group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE. CONCLUSIONS: We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2+TNF-α+Th2+ triple-positive and TNF-α+Th2+ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and the disease outcome in terms of control and persistence.

Assessment of CD27 expression as a tool for active and latent tuberculosis diagnosis.

UNLABELLED: There are still no reliable tests to distinguish active tuberculosis (TB) from latent TB infection (LTBI). Assessment of CD27 modulation on CD4⁺ T-cells has been suggested as a tool to diagnose different TB stages. OBJECTIVES: To use several cytometric approaches to evaluate CD27 expression on Mycobacterium tuberculosis (Mtb)-specific CD4⁺ T-cells to differentiate TB stages. METHODS: 55 HIV-uninfected subjects were enrolled: 13 active TB; 12 cured TB; 30 LTBI. Whole blood was stimulated with RD1-proteins or Cytomegalovirus-lysate (CMV). Interferon (IFN)-γ response was evaluated by cytometry. The proportion of CD27(±) within the IFN-γ⁺ CD4⁺ T-cells or RATIO of the CD27-median fluorescence intensity (MFI) of CD4⁺ T-cells over the CD27 MFI of IFN-γ⁺ CD4⁺ T-cells was evaluated. RESULTS: The greatest diagnostic accuracy in discriminating active TB vs. LTBI or cured TB was reached by evaluating the CD27(+) CD45RA(-) cells within the IFN-γ⁺ CD4⁺ T-cell subset (76.92 sensitivity for both, and 90% and 91.67% specificity, respectively), although the use of the CD27 MFI RATIO allows for stricter data analysis, independent of the operator. CONCLUSIONS: the study of CD27 expression using different approaches, whether it involves evaluation of CD45RA expression or not, is a robust biomarker for discriminating TB stages.

IL-4 specific-response in whole blood associates with human Cystic Echinococcosis and cyst activity.

OBJECTIVES: Human Cystic Echinococcosis (CE) is estimated in 2-3 million global cases. CE diagnosis and clinical management are based on imaging and serology, which lacks sensitivity and does not provide cyst stage information. This study aimed to evaluate tools for improving diagnosis by analysing the Interleukin (IL)-4-response to Antigen B (AgB) of Echinococcus granulosus. METHODS: Whole blood (WB) and peripheral blood mononuclear cells were stimulated with AgB. IL-4 levels were measured by enzyme-linked immunosorbent assay. RESULTS: WB 1-day stimulation resulted the best experimental condition for evaluating AgB IL-4-response. IL-4 levels were significantly higher in CE patients than healthy donors (p ≤ 0.0001). A ROC analysis showed significant area under the curve (AUC) results (AUC, 0.85; p = 0.0001) identifying an IL-4 level cut-off point ≥0.39 pg/mL which predicted CE with 71.4% sensitivity and 93.3% specificity. Moreover, we found that IL-4 levels were significantly increased in patients with active cysts compared to those with inactive cysts (p ≤ 0.0001). ROC analysis showed significant AUC results (0.94; p = 0.0001) with a cut-off point of 4.6 pg/mL which predicted active cysts with 84.6% sensitivity and 92% specificity. CONCLUSIONS: We found immunological correlates associated with CE and biological cyst activity.

Polyfunctional T-cells and effector memory phenotype are associated with active TB in HIV-infected patients.

OBJECTIVES: Polyfunctional T-cells associate with chronic viral infection control while their involvement in tuberculosis (TB) is unclear. We evaluated TB-specific polyfunctional T-cell response and memory status in antiretroviral treatment (ART)-naïve HIV-infected patients from a low TB-endemic country. METHODS: We prospectively enrolled HIV-infected patients, 12 with active TB (HIV-TB) and 15 with latent tuberculosis infection (LTBI). Peripheral blood cells were stimulated with TB antigens (RD1 proteins/peptides), HIV antigens, cytomegalovirus (CMV) and staphylococcal enterotoxin B (SEB) and analyzed by cytometry. RESULTS: The HIV-TB showed a higher frequency of polyfunctional CD4(+) T-cells in response to RD1 antigens than HIV-LTBI (p = 0.007). Among the CD8(+) T-cells, both groups showed a significantly higher frequency of RD1-specific monofunctional cells than polyfunctional cells (p = 0.03). Analyzing the cytokine profile, IFNγ(+) TNFα(+) CD4(+) T-cells associated with HIV-TB (p ≤ 0.02) whereas IL2(+) TNFα(+) associated with HIV-LTBI (p = 0.009). CD4(+) T-cell response presented an effector-memory status in HIV-TB (p = 0.007) and an effector-memory terminally-differentiated phenotype in HIV-LTBI (p = 0.03). CD8(+) T-cell response presented an effector status in HIV-LTBI (p = 0.02). No significant cytokine profile pattern associated with responses to the other stimuli tested. CONCLUSIONS: In HIV-infection, polyfunctional CD4(+) T-cell-response associates with active TB, characterized by a high proportion of IFNγ(+) TNFα(+) and an effector-memory phenotype.

IFNγ/TNFα specific-cells and effector memory phenotype associate with active tuberculosis.

OBJECTIVES: Controversial results were reported on the role of polyfunctional T-cells in tuberculosis (TB). Our aim was to simultaneously characterize the Mycobacterium tuberculosis (Mtb)-specific immune response as cytokine production and memory phenotype by flow cytometry after in vitro stimulation with region of difference 1 ("RD1") recombinant proteins (ESAT-6 and CFP-10) in patients at different TB stage in a low TB endemic country. To assess the specificity of these findings, we evaluated the response to cytomegalovirus (CMV), an unrelated antigen. METHODS: We enrolled subjects with active TB, cured TB, latent TB infection (LTBI). Cytokine and phenotype profiles of T-cells from whole blood stimulated with "RD1" proteins and CMV were characterized by multi-parametric flow cytometry. RESULTS: Bifunctional IFNγ(+) TNFα(+) CD4(+) T-cells and effector memory phenotype significantly associated with active TB compared to the LTBI group (p = 0.008, at least p ≤ 0.009 respectively) whereas "RD1"-T-cell response in cured TB and LTBI was characterized by a central memory phenotype (at least p ≤ 0.013 and p ≤ 0.004 respectively vs active TB). In contrast, response to CMV antigen was not associated with a TB-specific status. CONCLUSION: We identified qualitative associations between Mtb-specific T-cell and TB status in terms of functional capacity and memory status. These immune correlates may be helpful to trace natural history of TB.

Specific T cells restore the autophagic flux inhibited by Mycobacterium tuberculosis in human primary macrophages.

BACKGROUND: Autophagy inhibits survival of intracellular Mycobacterium tuberculosis when induced by rapamycin or interferon γ (IFN-γ), but it remains unclear whether M. tuberculosis itself can induce autophagy and whether T cells play a role in M. tuberculosis-mediated autophagy. The aim of this study was to evaluate the impact of M. tuberculosis on autophagy in human primary macrophages and the role of specific T cells in this process. METHODS: M. tuberculosis (H37Rv)-infected macrophages were incubated with naive or M. tuberculosis-specific T cells. Autophagy was evaluated at 4 hours and 8 hours after infection by analyzing the levels of LC3-II (a hallmark of autophagy) and p62 (a protein degraded by autophagy). M. tuberculosis survival was evaluated by counting the colony-forming units. RESULTS: M. tuberculosis infection of macrophages inhibited the autophagic process at 8 hours after infection. Naive T cells could not rescue this block, whereas M. tuberculosis-specific T cells restored autophagy degradation, accompanied by enhanced bacterial killing. Notably, the effect of M. tuberculosis-specific T cells was not affected by neutralization of endogenous IFN-γ and tumor necrosis factor α and was blocked by preventing contact between macrophages and T cells, suggesting that cell-cell interaction is crucial. CONCLUSIONS: M. tuberculosis inhibits autophagy in human primary macrophages, and specific T cells can restore functional autophagic flux through cell-cell contact.

Bystander inhibition of dendritic cell differentiation by Mycobacterium tuberculosis-induced IL-10.

Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1ß, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.