Novel insights in occupational asthma due to persulfate salts

El asma relacionada con el trabajo se define como el asma causada por la exposición a agentes que se encuentran en el lugar de trabajo, y es responsable de hasta un 25% de los casos de asma de inicio en la edad adulta. En los países industrializados es una de las causas más comunes de enfermedad respiratoria ocupacional. Concretamente, el asma ocupacional (AO) se atribuye a exposiciones en un particular ambiente ocupacional y no a estímulos fuera del lugar de trabajo, y puede estar inducido tanto por la sensibilización a un agente específico (AO inmunológico) como por exposición a sustancias irritantes (AO no inmunológico). Se han descrito más de 400 agentes causantes de AO y se dividen en agentes biológicos de alto peso molecular (APM) como proteínas, glicoproteínas y polisacáridos, y agentes químicos de bajo peso molecular (BPM) como sustancias químicas sintéticas, compuestos naturales, fármacos y metales. Las sales de persulfato son agentes químicos de BPM presentes en los productos decolorantes del cabello a concentraciones superiores al 60%. Estas sustancias son capaces de producir una sensibilización inmunológica y posterior enfermedad alérgica (dermatitis de contacto y asma bronquial), y son reconocidas como la principal causa de AO entre los profesionales de peluquería. Sin embargo, los detalles de la respuesta inmune implicada en el AO inducido por sales de persulfato no son bien conocidos, ya que parecen diferir de la respuesta típicamente alérgica de tipo 2. En algunos casos, se ha propuesto un mecanismo inmunológico mediado por inmunoglobulina-E (IgE) a pesar de las evidencias de una respuesta inmune tipo 1. En este sentido, un modelo murino de asma inducido por sales de persulfato, previamente validado, ha permitido ampliar el conocimiento de la fisiopatología implicada en este tipo de AO. La primera parte de la presente tesis se centra en el estudio de la persistencia de la respuesta asmática a sales de persulfato tras la sensibilización dérmica y tras una inhalación intranasal de persulfato amónico en ratones sensibilizados. Estos estudios mostraron una progresiva disminución de la respuesta asmática con el tiempo y los síntomas asmáticos llegaron incluso a desparecer, de forma parecida a lo que puede ocurrir en los pacientes con AO cuando cesa la exposición al agente causal. Sin embargo, no está claro que la completa eliminación de la exposición al agente sensibilizante sea la medida más eficiente ya que muchos pacientes permanecen sintomáticos. En este sentido, los ratones sensibilizados mostraron signos de sensibilización sistémica a largo plazo que les haría susceptibles a desarrollar una nueva respuesta asmática en el caso de una posible re-exposición al agente causal. El objetivo de la segunda parte de la tesis es evaluar el papel de la IgE y los mecanismos implicados en el desarrollo de la respuesta inmune en el AO causado por agentes de BPM, ya que el rol que desarrolla la IgE en este tipo de asma no está bien dilucidado. Mediante el bloqueo de la IgE, se pudieron estudiar los efectos del tratamiento con el anticuerpo monoclonal (AcM) anti-IgE en el modelo murino de AO inducido por sales de persulfato. La administración del AcM anti-IgE neutralizó completamente los niveles de IgE en suero y mejoró los síntomas asmáticos como la hiperrespuesta bronquial y los parámetros inflamatorios. Estos hallazgos sugieren la implicación de un mecanismo inmunológico donde la IgE puede tener un papel relevante para la fisiopatología del asma causada por agentes de BPM. En conclusión, los estudios de esta tesis arrojan conocimientos en la fisiopatología del AO a sales de persulfato y proponen una compleja interacción entre la respuesta inmune innata y una respuesta adaptativa mixta tipo1-tipo2

Persistence of Asthmatic Response after Ammonium Persulfate-Induced Occupational Asthma in Mice

Since persulfate salts are an important cause of occupational asthma (OA), we aimed to study the persistence of respiratory symptoms after a single exposure to ammonium persulfate (AP) in AP-sensitized mice. BALB/c mice received dermal applications of AP or dimethylsulfoxide (DMSO) on days 1 and 8. On day 15, they received a single nasal instillation of AP or saline. Airway hyperresponsiveness (AHR) was assessed using methacholine provocation, while pulmonary inflammation was evaluated in bronchoalveolar lavage (BAL), and total serum immunoglobulin E (IgE), IgG1 and IgG2a were measured in blood at 1, 4, 8, 24 hours and 4, 8, 15 days after the single exposure to the causal agent. Histological studies of lungs were assessed. AP-treated mice showed a sustained increase in AHR, lasting up to 4 days after the challenge. There was a significant increase in the percentage of neutrophils 8 hours after the challenge, which persisted for 24 hours in AP-treated mice. The extent of airway inflammation was also seen in the histological analysis of the lungs from challenged mice. Slight increases in total serum IgE 4 days after the challenge were found, while IgG gradually increased further 4 to 15 days after the AP challenge in AP-sensitized mice. In AP-sensitized mice, an Ig-independent response is induced after AP challenge. AHR appears immediately, but airway neutrophil inflammation appears later. This response decreases in time; at early stages only respiratory and inflammatory responses decrease, but later on immunological response decreases as well

Persistence of respiratory and inflammatory responses after dermal sensitization to persulfate salts in a mouse model of non-atopic asthma

Exposure to ammonium persulfate (AP) has been reported to be the main cause of occupational asthma in hairdressers. The aim of this study is to assess how long the asthmatic response to AP can be induced after dermal sensitization in a mouse model. BALB/c mice received dermal applications of AP or dimethylsulfoxide (DMSO) (control) on days 1 and 8. They then received a single nasal instillation (challenge) of AP or saline on days 15, 22, 29, 36, 45, 60 and 90. Respiratory responsiveness to methacholine was measured 24 h after the challenge using a non-specific methacholine provocation test. Pulmonary inflammation was analysed in bronchoalveolar lavage (BAL), and total serum immunoglobulin (Ig) E, IgG1 and IgG2a were measured in serum samples. Histological analysis of lung slides was performed. Mice dermally sensitized and intranasally challenged with AP showed respiratory responsiveness to methacholine as long as 45 days after initial sensitization, as well as increased percentage of neutrophils in BAL compared with the control group. At day 60, dermally sensitized mice still presented bronchial hyperresponsiveness, while the percentage of neutrophils returned to baseline levels similar to those of controls. Total serum IgE increased significantly on day 22 after dermal sensitization. Total serum IgG1 and IgG2a increased from 45 days after dermal sensitization and remained high at 90 days. Both respiratory responsiveness to methacholine and airway inflammation responses decrease with increasing time between sensitization and challenge. Respiratory responsiveness to methacholine tends to persist longer than inflammation

Novel insights in occupational asthma due to persulfate salts

El asma relacionada con el trabajo se define como el asma causada por la exposición a agentes que se encuentran en el lugar de trabajo, y es responsable de hasta un 25% de los casos de asma de inicio en la edad adulta. En los países industrializados es una de las causas más comunes de enfermedad respiratoria ocupacional. Concretamente, el asma ocupacional (AO) se atribuye a exposiciones en un particular ambiente ocupacional y no a estímulos fuera del lugar de trabajo, y puede estar inducido tanto por la sensibilización a un agente específico (AO inmunológico) como por exposición a sustancias irritantes (AO no inmunológico). Se han descrito más de 400 agentes causantes de AO y se dividen en agentes biológicos de alto peso molecular (APM) como proteínas, glicoproteínas y polisacáridos, y agentes químicos de bajo peso molecular (BPM) como sustancias químicas sintéticas, compuestos naturales, fármacos y metales. Las sales de persulfato son agentes químicos de BPM presentes en los productos decolorantes del cabello a concentraciones superiores al 60%. Estas sustancias son capaces de producir una sensibilización inmunológica y posterior enfermedad alérgica (dermatitis de contacto y asma bronquial), y son reconocidas como la principal causa de AO entre los profesionales de peluquería. Sin embargo, los detalles de la respuesta inmune implicada en el AO inducido por sales de persulfato no son bien conocidos, ya que parecen diferir de la respuesta típicamente alérgica de tipo 2. En algunos casos, se ha propuesto un mecanismo inmunológico mediado por inmunoglobulina-E (IgE) a pesar de las evidencias de una respuesta inmune tipo 1. En este sentido, un modelo murino de asma inducido por sales de persulfato, previamente validado, ha permitido ampliar el conocimiento de la fisiopatología implicada en este tipo de AO. La primera parte de la presente tesis se centra en el estudio de la persistencia de la respuesta asmática a sales de persulfato tras la sensibilización dérmica y tras una inhalación intranasal de persulfato amónico en ratones sensibilizados. Estos estudios mostraron una progresiva disminución de la respuesta asmática con el tiempo y los síntomas asmáticos llegaron incluso a desparecer, de forma parecida a lo que puede ocurrir en los pacientes con AO cuando cesa la exposición al agente causal. Sin embargo, no está claro que la completa eliminación de la exposición al agente sensibilizante sea la medida más eficiente ya que muchos pacientes permanecen sintomáticos. En este sentido, los ratones sensibilizados mostraron signos de sensibilización sistémica a largo plazo que les haría susceptibles a desarrollar una nueva respuesta asmática en el caso de una posible re-exposición al agente causal. El objetivo de la segunda parte de la tesis es evaluar el papel de la IgE y los mecanismos implicados en el desarrollo de la respuesta inmune en el AO causado por agentes de BPM, ya que el rol que desarrolla la IgE en este tipo de asma no está bien dilucidado. Mediante el bloqueo de la IgE, se pudieron estudiar los efectos del tratamiento con el anticuerpo monoclonal (AcM) anti-IgE en el modelo murino de AO inducido por sales de persulfato. La administración del AcM anti-IgE neutralizó completamente los niveles de IgE en suero y mejoró los síntomas asmáticos como la hiperrespuesta bronquial y los parámetros inflamatorios. Estos hallazgos sugieren la implicación de un mecanismo inmunológico donde la IgE puede tener un papel relevante para la fisiopatología del asma causada por agentes de BPM. En conclusión, los estudios de esta tesis arrojan conocimientos en la fisiopatología del AO a sales de persulfato y proponen una compleja interacción entre la respuesta inmune innata y una respuesta adaptativa mixta tipo1-tipo2.The exposure to specific agents present in the workplace is thought to account for up to 25% of all cases of adult-onset asthma leading to work-related asthma, which is a common cause of work-related lung disease in the industrial world. Specifically, occupational asthma (OA) is attributable to exposure in a particular work environment and not to stimuli outside the workplace, induced by either sensitization to a specific substance (sensitizer-induced OA) or by exposure to an inhaled irritant at work (irritant-induced OA). More than 400 agents are reported to cause OA. They can be divided into biological agents of high molecular weight (HMW) (>5 KDa), such as proteins, glycoproteins and polysaccharides, and chemical agents of low molecular weight (LMW) (< 5 KDa) such as synthetic chemicals, natural compounds, drugs and metals. Persulfate salts are LMW chemical compounds present in hair bleaching products at concentrations up to 60%. They are capable of causing immunological sensitization and subsequent allergic disease (such as contact dermatitis and bronchial asthma), and are the main cause of OA among hairdressers. Nevertheless, no consensus has been reached regarding the details of the immune response involved in persulfate-induced OA, as it seems to differ from the typical allergic type2 immune response. In some cases, an IgE-mediated mechanism has been proposed despite evidence of a type 1 immune response. In this connection, a validated mouse model of persulfate-induced asthma has provided valuable knowledge about the physiopathology involved in this type of OA. The first part of this thesis focuses on the study of the persistence of the asthmatic response to persulfate salts after dermal sensitization (Chapter 1) and after specific persulfate challenge in sensitized mice (Chapter 2) in the mouse model of persulfate-induced asthma. These studies showed a progressive decrease in the asthmatic response over time and even found that the asthma symptoms may disappear, perhaps mirroring what happens in patients when the exposure to the causal agent ceases. Nevertheless, it is not clear that complete removal from the exposure to the sensitizing agent is the most efficient therapeutic approach, as many patients remain symptomatic despite avoidance of the causal agent. In this context, sensitized mice exhibited signs of long-term sensitization which would make them susceptible to developing a new asthmatic response when re-exposed to the sensitizing agent. The aim of the second part of this thesis was to explore the role of IgE and the mechanisms involved in the development of the immune response in this type of OA due to LMW agents. By neutralizing the IgE, we wanted to study the effects of anti-IgE monoclonal antibody (mAb) treatment in the established mouse model of persulfate-induced OA (Chapter 3). The administration of anti-IgE mAb completely neutralized serum IgE and improved asthma symptoms such as airway hyperresponsiveness (AHR) and inflammation patterns in the mouse model of OA, suggesting that an immunological mechanism is involved and that IgE may play an important role in the pathophysiology of the chemical-induced asthma. In conclusion, the studies included in his doctoral thesis shed light on the pathophysiology of OA due to persulfate salts and propose a complex interaction of innate and a mixed type1-type2 adaptative immune response

Persistence of asthmatic response after ammonium persulfate-induced occupational asthma in mice

Since persulfate salts are an important cause of occupational asthma (OA), we aimed to study the persistence of respiratory symptoms after a single exposure to ammonium persulfate (AP) in AP-sensitized mice.status: publishe

Persistence of asthmatic response after ammonium persulfate-induced occupational asthma in mice.

IntroductionSince persulfate salts are an important cause of occupational asthma (OA), we aimed to study the persistence of respiratory symptoms after a single exposure to ammonium persulfate (AP) in AP-sensitized mice.Material and methodsBALB/c mice received dermal applications of AP or dimethylsulfoxide (DMSO) on days 1 and 8. On day 15, they received a single nasal instillation of AP or saline. Airway hyperresponsiveness (AHR) was assessed using methacholine provocation, while pulmonary inflammation was evaluated in bronchoalveolar lavage (BAL), and total serum immunoglobulin E (IgE), IgG1 and IgG2a were measured in blood at 1, 4, 8, 24 hours and 4, 8, 15 days after the single exposure to the causal agent. Histological studies of lungs were assessed.ResultsAP-treated mice showed a sustained increase in AHR, lasting up to 4 days after the challenge. There was a significant increase in the percentage of neutrophils 8 hours after the challenge, which persisted for 24 hours in AP-treated mice. The extent of airway inflammation was also seen in the histological analysis of the lungs from challenged mice. Slight increases in total serum IgE 4 days after the challenge were found, while IgG gradually increased further 4 to 15 days after the AP challenge in AP-sensitized mice.ConclusionsIn AP-sensitized mice, an Ig-independent response is induced after AP challenge. AHR appears immediately, but airway neutrophil inflammation appears later. This response decreases in time; at early stages only respiratory and inflammatory responses decrease, but later on immunological response decreases as well

Levels of interleukin (IL)-2, IL-10 and IL-13 in BAL fluid.

<p>BAL samples were collected 1, 4, 8 and 24 hours, and 4, 8 and 15 days after AP challenge. Experimental groups are the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109000#pone-0109000-g001" target="_blank">figure 1</a>. <b>A</b>) Mean ± SEM of IL-13 concentration. <b>B</b>) Mean ± SEM of IL-10 concentration. <b>C</b>) Mean ± SEM of IL-2 concentration. Ψ: p = 0.083 compared with DMSO/SAL, π: p = 0.053 compared with DMSO/SAL, Ω: p = 0.055 compared with DMSO/AP, ξ: p = 0.076 compared with DMSO/SAL. No significant differences were found in the other groups studied at different time intervals. AP, ammonium persulfate; BAL, bronchoalveolar lavage; DMSO, dimethylsulfoxide; IL, interleukin; SAL, saline.</p

Airway hyperresponsiveness (AHR) to methacholine expressed as resistance (R) was measured 1 hour, 4 hours, 8 hours, 24 hours, 4 days, 8 days and 15 days after intranasal instillation by the forced oscillation technique to increasing concentrations of methacholine.

<p>Experimental groups were DMSO/SAL, DMSO/AP and AP/AP. First abbreviation refers to dermal sensitization (day 1 and 8), and the second to the agents administered via intranasal instillation (day 15). <b>A</b>) Mean ± SEM of AUC of R against methacholine concentrations (0 to 10 mg/ml) for all periods of time. <b>B</b>) Mean individual values of AUC 1 hour, 24 hours, 4 days and 15 days after challenge. *p<0.05, **p<0.01 compared with DMSO/SAL, ++p<0.01 compared with DMSO/AP, Ψp<0.05 and ΨΨp<0.01 when DMSO/SAL is compared with DMSO/AP. No significant differences were found in the other groups studied at different time intervals. AP, ammonium persulfate; AUC, area under the curve; DMSO, dimethylsulfoxide; SAL, saline.</p

Lung histopathology.

<p>Representative images of lung sections are shown at low and high magnification. <b>A</b>) Haematoxylin and eosin stained histological lung sections. <b>B</b>) Massons's trichrome stained histological lung sections. Experimental groups in this figure are represented with sections from DMSO/SAL, DMSO/AP groups and AP/AP groups assessed 8 hours and 4 days after AP challenge. AP, ammonium persulfate, DMSO, dimethylsulfoxide; SAL, saline.</p

Total number of macrophages (A), neutrophils (B) and lymphocytes (C) in BAL obtained 1, 4, 8 and 24 hours, and 4, 8 and 15 days after AP challenge.

<p>Experimental groups are the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109000#pone-0109000-g001" target="_blank">figure 1</a>. Mean ± SEM of total number of neutrophils in BAL. *p<0.05 compared with DMSO/SAL, +p<0.05 compared with DMSO/PA. No significant differences were found in the other groups studied at different time points. AP, ammonium persulfate, BAL, bronchoalveolar lavage, DMSO, dimethylsulfoxide; SAL, saline.</p