Aaron A. Berlin

Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2(-/-) mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.

AMD3100, a CxCR4 Antagonist, Attenuates Allergic Lung Inflammation and Airway Hyperreactivity

The role of specific chemokine receptors during allergic asthmatic responses has been relatively undefined. A number of receptors are preferentially expressed on Th2 cells, including CCR4, CCR8, and CxCR4. In the present study, we have examined the role of CxCR4 in the development of cockroach allergen-induced inflammation and airway hyperreactivity in a mouse model of asthma. Using a specific inhibitor of CxCR4, AMD3100, our results indicate that blocking this receptor has a significant effect in down-regulating the inflammation and pathophysiology of the allergen-induced response. Treatment of allergic mice with AMD3100 significantly reduced airway hyperreactivity, peribronchial eosinophilia, and the overall inflammatory responses. In addition, there was a shift in the cytokine profile that was observed in the AMD3100-treated animals. Specifically, there was a significant reduction in interleukin-4 and interleukin-5 levels and a significant increase in interleukin-12 and interferon-gamma levels within the lungs of treated allergic mice. Furthermore, there was a significant alteration in the local chemokine production of CCL22 (MDC) and CCL17 (TARC), two chemokines previously shown to be important in Th2-type allergen responses. Overall, specifically blocking CxCR4 using AMD3100 reduced a number of pathological parameters related to asthmatic-type inflammation.

Deletion of TLR3 Alters the Pulmonary Immune Environment and Mucus Production during Respiratory Syncytial Virus Infection

The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity. In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion. Although we observed no changes in viral growth, we did find that TLR3−/− mice demonstrated significant increases in mucus production in the airways of RSV-infected mice. The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein. The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression. Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3−/− mice. Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3−/− mice, but not wild-type mice, after RSV infection. Isolated lung draining lymph node cells from TLR3−/− mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection. To demonstrate a causative link, we depleted TLR3−/− mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated. Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease.

IL-13-Induced Airway Hyperreactivity During Respiratory Syncytial Virus Infection Is STAT6 Dependent

Airway damage and hyperreactivity induced during respiratory syncytial virus (RSV) infection can have a prolonged effect in infants and young children. These infections can alter the long-term function of the lung and may lead to severe asthma-like responses. In these studies, the role of IL-13 in inducing and maintaining a prolonged airway hyperreactivity response was examined using a mouse model of primary RSV infection. Using this model, there was evidence of significant airway epithelial cell damage and sloughing, along with mucus production. The airway hyperreactivity response was significantly increased by 8 days postinfection, peaked during days 10–12, and began to resolve by day 14. When the local production of Th1- and Th2-associated cytokines was examined, there was a significant increase, primarily in IL-13, as the viral response progressed. Treatment of RSV-infected mice with anti-IL-13 substantially inhibited airway hyperreactivity. Anti-IL-4 treatment had no effect on the RSV-induced responses. Interestingly, when IL-13 was neutralized, an early increase in IL-12 production was observed within the lungs, as was a significantly lower level of viral Ags, suggesting that IL-13 may be regulating an important antiviral pathway. The examination of RSV-induced airway hyperreactivity in STAT6−/− mice demonstrated a significant attenuation of the response, similar to the anti-IL-13 treatment. In addition, STAT6−/− mice had a significant alteration of mucus-producing cells in the airway. Altogether, these studies suggest that a primary factor leading to chronic RSV-induced airway dysfunction may be the inappropriate production of IL-13.

Respiratory Syncytial Virus Predisposes Mice to Augmented Allergic Airway Responses Via IL-13-Mediated Mechanisms

The development of severe childhood asthma may be influenced by several factors including environmental and infectious stimuli. The causal relationship between infectious viral responses, such as respiratory syncytial virus (RSV), and severe asthma during early childhood is unclear. In these studies, the ability for an initial RSV infection to exacerbate and promote a more severe asthmatic-type response was investigated by combining established murine models of disease. We examined the ability of RSV to induce exacerbation of allergic disease over a relatively long period, leading to development of severe airway responses including airway inflammation and hyperreactivity. The preferential production of IL-13 during a primary RSV infection appears to play a critical role for the exacerbation of cockroach allergen-induced disease. The depletion of IL-13 during RSV infections inhibited the exacerbation and acceleration of severe allergen-induced airway hyperreactivity. This was indicated by decreases in airway hyperreactivity and changes in lung chemokine production. These data suggest that the airway responses during asthma can be greatly affected by a previous RSV infection, even when infection occurs before allergen sensitization. Overall, infection of the airways with RSV can induce an IL-13-dependent change in airway function and promotes an environment that contributes to the development of severe allergic asthmatic responses.

A selective novel low‐molecular‐weight inhibitor of IκB kinase‐β (IKK‐β) prevents pulmonary inflammation and shows broad anti‐inflammatory activity

1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell‐mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF‐κB). 3 NF‐κB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK‐β. 4 We identified COMPOUND A as a small‐molecule, ATP‐competitive inhibitor selectively targeting IKK‐β kinase activity with a Ki value of 2 nM. 5 COMPOUND A inhibited stress‐induced NF‐κB transactivation, chemokine‐, cytokine‐, and adhesion molecule expression, and T‐ and B‐cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS‐induced TNF‐α in rodents. 7 In mice COMPOUND A inhibited cockroach allergen‐induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed‐type hypersensitivity. 10 These data suggest that IKK‐β inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.

IL-17–Induced Pulmonary Pathogenesis during Respiratory Viral Infection and Exacerbation of Allergic Disease

Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17(-/-) knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections.

Thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to infection.

BACKGROUND Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis and asthma exacerbation. OBJECTIVE The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection. METHODS Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air-liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection. RESULTS Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-κB. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology. CONCLUSION These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.

Respiratory syncytial virus-induced CCL5/RANTES contributes to exacerbation of allergic airway inflammation

Severe respiratory syncytial virus (RSV) infection has a significant impact on airway function and may induce or exacerbate the response to a subsequent allergic challenge. In a murine model combining early RSV infection with later cockroach allergen (CRA) challenge, we examined the role of RSV‐induced CCL5/RANTES production on allergic airway responses. RSV infection increased CCL5 mRNAand protein levels, peaking at days 8 and 12, respectively. Administration of CCL5 antiserum during days 0–14 of the RSV infection did not significantly alter viral protein expression when comparedto mice treated with control serum. In mice receiving the combined RSV‐allergen challenge, lungs collected on day 22 exhibited significantly increased numbers of CD4‐ and CD8‐positive T cells. Thisincrease in T cell numbers was not observed in mice receiving α‐CCL5. On day 43, peribronchial eosinophilia and leukotriene levels were increased in RSV‐allergen mice. Pretreatment with CCL5 antiserum resulted in decreased recruitment of inflammatory cells to bronchoalveolar and peribronchial regions of the lungs and these reductions were associated with a reduction in both T cell recruitment into the bronchoalveolar space, leukotriene release and chemokine generation. Thus, CCL5 released during RSV infection has a significant effect on the inflammatory response to subsequent allergic airway challenges.

Rantes (CCL5) production during primary respiratory syncytial virus infection exacerbates airway disease

Respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity in infants and young children. The importance of chemokines during RSV infection for respiratory symptoms has not been fully elucidated. The current study examined the effect of RANTES (CCL5) on airway pathophysiology after RSV infection. BALB/c mice produce RANTES (CCL5) after RSV infection that correlates with the changes in pathophysiology. Animals treated with anti‐RANTES (CCL5) antibody demonstrated significant decreases in airway hyperreactivity (AHR). Delayed treatment with anti‐RANTES (CCL5) at day 5 of infection also significantly reduced development of AHR on day 9 of infection, suggesting that RANTES (CCL5) may be a target in established disease. Determination of Th1/Th2‐associated cytokine patterns indicated that anti‐RANTES (CCL5) treatment increased IL‐12 production, thus altering the lung environment. The assessment of RANTES (CCL5) production in vitro and in vivo demonstrated that it was regulated by IL‐13, a cytokine that is related to RSV‐induced AHR in this mouse model. These data show that RANTES (CCL5) is an important mediator of the pathophysiological responses seen in RSV infection.