COPD Patients Exhibit Distinct Gene Expression, Accelerated Cellular Aging, and Bias to M2 Macrophages.

COPD, one of world's leading contributors to morbidity and mortality, is characterized by airflow limitation and heterogeneous clinical features. Three main phenotypes are proposed: overlapping asthma/COPD (ACO), exacerbator, and emphysema. Disease severity can be classified as mild, moderate, severe, and very severe. The molecular basis of inflammatory amplification, cellular aging, and immune response are critical to COPD pathogenesis. Our aim was to investigate EP300 (histone acetylase, HAT), HDAC 2 (histone deacetylase), HDAC3, and HDAC4 gene expression, telomere length, and differentiation ability to M1/M2 macrophages. For this investigation, 105 COPD patients, 42 smokers, and 73 non-smoker controls were evaluated. We identified a reduced HDAC2 expression in patients with mild, moderate, and severe severity; a reduced HDAC3 expression in patients with moderate and severe severity; an increased HDAC4 expression in patients with mild severity; and a reduced EP300 expression in patients with severe severity. Additionally, HDAC2 expression was reduced in patients with emphysema and exacerbator, along with a reduced HDAC3 expression in patients with emphysema. Surprisingly, smokers and all COPD patients showed telomere shortening. COPD patients showed a higher tendency toward M2 markers. Our data implicate genetic changes in COPD phenotypes and severity, in addition to M2 prevalence, that might influence future treatments and personalized therapies.

Impact of JAK/STAT inhibitors on human monocyte-derived-macrophages stimulated by cigarette smoke extract and lipopolysaccharide.

The main risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoke (CS). It can alter many immune cells functions such as phagocytosis, efferocytosis and cytokine production. Cytokines play a role in the orchestration of inflammation in COPD. The JAK/STAT pathways are among the most important signalling components of cytokines. The objective of this work was to investigate the role of the JAK/STAT pathway with regard to cytokine release and microsphere uptake capacity (to minimize the non-specific scavenging) in human monocyte-derived-macrophages (MDMs). The MDMs were stimulated by cigarette smoke extract (CSE) alone or in combination with lipopolysaccharide (LPS). CSE alone was not associated with significant changes in the cytokine, with the exception of IL-8/CXCL8 production. However, CSE disturbed cytokine production in LPS-stimulated MDMs. CSE increase CXCL-8 and CCL2 release in LPS-stimulated monocyte-derived macrophages and suppressed the production of IL-6 and CXCL1 in these cells. CSE also decreased microsphere uptake capacity by MDMs. Then, CSE + LPS-stimulated MDMs were treated with two different JAK inhibitors. AG490 (specific inhibitor of JAK2) and ruxolitinib (inhibitor of JAK1 and JAK2). JAK/STAT inhibitors, particularly ruxolitinib, attenuated in cytokine production without completely inhibiting when compared with dexamethasone. On the other hand, the cells exposed to dexamethasone are nearly unable to capture the microspheres, while both JAK inhibitors do not affect the uptake capacity. In summary, our results showed the versatility of ruxolitinib which might bring a better balance disturbance of cytokine release and uptake capacity. The information regarding the distinctive effect of JAK/STAT inhibitors may be useful in the development of novel treatments for COPD.

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells.

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human ß-defensin-2 (hßD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.

Alteration of immunophenotype of human macrophages and monocytes after exposure to cigarette smoke.

Cigarette smoke exposure (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Macrophages have an important role in COPD because they release pro-inflammatory and anti-inflammatory cytokines. The present study's we investigate the functional changes in macrophages and monocytes exposed to cigarette smoke extract (CSE). Herein, using human monocyte-derived macrophages (MDMs) from healthy donors and we found that CSE was not associated with significant changes in the production of pro inflammatory cytokines by MDMs. In contrast, exposure to CSE suppressed the production of IL-6 and Gro-a/CXCL1 by LPS-stimulated-MDMs, but had an additive effect on the release of IL-8/CXCL8 and MCP1/CCL2. However, CSE exposure was associated with greater production, TARC/CCL-17 and CCL22/MDC. Moreover, MDMs displayed a lower uptake capacity after CSE exposure. We identify, for what is to our knowledge the first time that monocytes from patients with COPD produced less IL-8/CXCL8 and Gro-α/CXCL1 after LPS stimulation and produced higher levels of TARC/CCL17 and MDC/CCL-22 after IL-4 stimulation. Our present results highlighted a skewed immune response, with an imbalance in M1 vs. M2 cytokine production. In conclusion, exposure to CS has contrasting, multifaceted effects on macrophages and monocytes. Our data may provide a better understanding of the mechanisms underlying COPD.

Conhecer para prevenir: complementação do conhecimento dos alunos do ensino fundamental sobre microbiologia e parasitologia em três escolas de Uberlândia-MG / Know to prevent: complementation of the knowledge of the students of fundamental education on microbiology and parasitology in three schools of Uberlândia-MG

O conhecimento sobre microbiologia e parasitologia é considerado abstrato, pois muitos agentes causadores de doenças não são vistos a olho nu, o que distancia os alunos da realidade. Assim, nossos objetivos foram promover ações educativas por meio de ferramentas didáticas lúdicas que possibilitassem a complementação do aprendizado dos alunos frente aos diferentes grupos de microorganismos e parasitos e das ações de profilaxia relacionadas aos mesmos, e avaliar se ao final eles tinham condições de discriminar os grupos e relacionar com as doenças e as formas de profilaxia. Para tanto, foram feitas entrevistas junto aos professores para levantamento das possíveis atividades a serem desenvolvidas. A ação foi definida e então dividida em três momentos (aula expositiva, jogo didático e mostra científica) realizados entre agosto e novembro de 2018, atingindo aproximadamente 350 alunos, de oitavos e nonos anos, de três escolas públicas da zona urbana e rural da cidade de Uberlândia-MG. Para avaliar o impacto da ação foi feita uma análise comparativa de questionários aplicados antes (pré-intervenção) e após a ação (pós-intervenção). O percentual das respostas corretas nos questionários pós-intervenção aumentou em duas escolas (p>0,005). Quanto à análise por questões, as menores porcentagens de acertos foram observadas em perguntas relacionadas à distinção entre doenças bacterianas e virais, o reconhecimento dos sintomas e a associação das formas de transmissão com a profilaxia. Assim, este estudo reforça a importância da educação em saúde para que os alunos se mobilizem frente ao combate das doenças.(AU)

The knowledge about microbiology and parasitology is considered abstract since causative agents of diseases cannot be seen with the naked eye, leading to students' detachment from reality. Therefore, this work aimed to promote educational actions through playful tools that could complement students' learning regarding the different groups of microorganisms and parasites and the prophylactic measures related to them. Furthermore, at the end of the actions it was evaluated if the students were able to discriminate the groups of microorganisms and relate them to the diseases they cause and the different forms of prophylaxis. To this end, interviews were conducted with teachers to survey the possible activities that could be used. The action was defined and then divided into three moments (expository class, didactic game and scientific show) held between August and November 2018, reaching approximately 350 students, from the eighth and ninth years, from three public schools in the urban and rural area of the city oftUberlândia-MG. To assess the impact of the action, a comparative analysis of questionnaires was applied before (pre-intervention) and after the action (post-intervention). The percentage of correct answers in the questionnaires post-intervention increased in two schools (p>0,005). Regarding the analysis by questions, the lowest percentages of correct answers were observed in questions related to the distinction between bacterial and viral diseases, the recognition of the symptoms and the association of transmission ways with prophylaxis. Thus, this study reinforces the importance of health education for students to mobilize in the fight against diseases.(AU)

Erythema Nodosum Leprosum Neutrophil Subset Expressing IL-10R1 Transmigrates into Skin Lesions and Responds to IL-10.

Erythema nodosum leprosum (ENL) is an inflammatory complication in leprosy. Yet, the involvement of ENL neutrophils in the inflammatory response against Mycobacterium leprae remains poorly explored. Our primary aim was to investigate the utility of the surface expression of neutrophil IL-10R1 as an ENL biomarker and, secondarily, to evaluate whether leprosy or healthy M. leprae-stimulated neutrophils produce cytokines and are able to respond to IL-10. We, in this study, describe a subpopulation of circulating neutrophils of ENL patients that exclusively expressed IL-10R1, providing evidence that IL-10R1+ neutrophils are present in ENL lesions. It was also found that ENL neutrophils, but not those of nonreactional leprosy controls, were able to secret detectable levels of TNF ex vivo and the addition of IL-10 blocked TNF release. It was likewise observed that M. leprae-stimulated, healthy neutrophils expressed IL-10R1 in vitro, and ENL-linked cytokines were released by M. leprae-cultured neutrophils in vitro. Moreover, consistent with the presence of a fully functional IL-10R, the addition of IL-10 prevented the release of M. leprae-induced cytokines. Most importantly, dead M. leprae revealed its superior capacity to induce CCL4 and IL-8 in primary neutrophils over live Mycobacterium, suggesting that M. leprae may hamper the inflammatory machinery as an immune escape mechanism.

Neutrophil extracellular traps contribute to the pathogenesis of leprosy type 2 reactions.

Up to 50% of patients with the multibacillary form of leprosy are expected to develop acute systemic inflammatory episodes known as type 2 reactions (T2R), thus aggravating their clinical status. Thalidomide rapidly improves T2R symptoms. But, due to its restricted use worldwide, novel alternative therapies are urgently needed. The T2R triggering mechanisms and immune-inflammatory pathways involved in its pathology remain ill defined. In a recent report, we defined the recognition of nucleic acids by TLR9 as a major innate immunity pathway that is activated during T2R. DNA recognition has been described as a major inflammatory pathway in several autoimmune diseases, and neutrophil DNA extracellular traps (NETs) have been shown to be a prime source of endogenous DNA. Considering that neutrophil abundance is a marked characteristic of T2R lesions, the objective of this study was to investigate NETs production in T2R patients based on the hypothesis that the excessive NETs formation would play a major role in T2R pathogenesis. Abundant NETs were found in T2R skin lesions, and increased spontaneous NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression was shown to be higher in T2R neutrophils, suggesting that DNA recognition via TLR9 may be one of the pathways triggering this process during T2R. Finally, treatment of T2R patients with thalidomide for 7 consecutive days resulted in a decrease in all of the evaluated in vivo and ex vivo NETosis parameters. Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, will contribute to the emergence of novel alternative therapies and the identification of prognostic reactional markers in the near future.

Neutrophils in Leprosy.

Leprosy is an infectious disease caused by the intracellular bacillus Mycobacterium leprae that mainly affects the skin and peripheral nerves. One of the most intriguing aspects of leprosy is the diversity of its clinical forms. Paucibacillary patients are characterized as having less than five skin lesions and rare bacilli while the lesions in multibacillary patients are disseminated with voluminous bacilli. The chronic course of leprosy is often interrupted by acute episodes of an inflammatory immunological response classified as either reversal reaction or erythema nodosum leprosum (ENL). Although ENL is considered a neutrophilic immune-complex mediated condition, little is known about the direct role of neutrophils in ENL and leprosy disease overall. Recent studies have shown a renewed interest in neutrophilic biology. One of the most interesting recent discoveries was that the neutrophilic population is not homogeneous. Neutrophilic polarization leads to divergent phenotypes (e.g., a pro- and antitumor profile) that are dynamic subpopulations with distinct phenotypical and functional abilities. Moreover, there is emerging evidence indicating that neutrophils expressing CD64 favor systemic inflammation during ENL. In the present review, neutrophilic involvement in leprosy is discussed with a particular focus on ENL and the potential of neutrophils as clinical biomarkers and therapeutic targets.

Elevated Pentraxin-3 Concentrations in Patients With Leprosy: Potential Biomarker of Erythema Nodosum Leprosum.

Background: Leprosy, the leading infectious cause of disability worldwide, remains a major public health challenge in the most severely affected countries despite the sharp decline in new cases in recent years. The search for biomarkers is essential to achieve a better understanding of the molecular and cellular mechanisms underlying the disease. Methods: Pentraxin-3 (PTX3) analyses of sera from 87 leprosy patients with or without reactions were conducted via enzyme-linked immunosorbent assay. In situ identification of PTX3 in skin lesion was confirmed by quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunofluorescence assays. Results: We found that PTX3 serum levels were higher in multibacillary patients when evaluated before the onset of acute erythema nodosum leprosum (ENL) and persistently elevated during reaction. Thalidomide treatment reduced PTX3 in the serum 7 days after starting treatment. In situ analyses have also demonstrated enhancement of PTX3 in ENL lesions and showed that treatment with thalidomide reduced its expression and the prominent neutrophilic infiltrate, a hallmark of the disease. Conclusions: In summary, our study provides in vivo evidence that PTX3 is enhanced during ENL but not in reversal reaction and provides a new molecular target in ENL pathogenesis.