Sudha Natarajan

Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo.

Chromatin‐modifying HDACi exhibit anti‐inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM‐CSF‐stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b+Ly6C+F4/80intCD115+), which suppressed allogeneic T cell proliferation in a NOS‐ and HO‐1‐dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin–Sca‐1+c‐Kit+). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM‐CSF administration and increased the number of CD11b+Gr1+ cells in BM and spleen. Increased numbers of Gr1+ cells, which suppressed T cell proliferation, were recovered from spleens of TSA‐treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti‐inflammatory action of HDACi.

Acute Pulmonary Lipopolysaccharide Tolerance Decreases TNF-α without Reducing Neutrophil Recruitment

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-α. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-α were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-α mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-α was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-α expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-α levels, while still maintaining the antimicrobial capacity of the lung.

Diesel Exhaust Particulates Exacerbate Asthma-Like Inflammation by Increasing CXC Chemokines

Particulate matter heavily pollutes the urban atmosphere, and several studies show a link between increased ambient particulate air pollution and exacerbation of pre-existing pulmonary diseases, including asthma. We investigated how diesel exhaust particulates (DEPs) aggravate asthma-like pulmonary inflammation in a mouse model of asthma induced by a house dust extract (HDE) containing cockroach allergens and endotoxin. BALB/c mice were exposed to three pulmonary challenges via hypopharyngeal administration of an HDE collected from the home of an asthmatic child. One hour before each pulmonary challenge, mice were exposed to DEP or PBS. Pulmonary inflammation was assessed by histological features, oxidative stress, respiratory physiological features, inflammatory cell recruitment, and local CXC chemokine production. To prove the role of CXC chemokines in the augmented inflammation, CXC chemokine-specific antibodies were delivered to the lungs before DEP exposure. DEP exacerbated HDE-induced airway inflammation, with increased airway mucus production, oxidative stress, inflammatory cell infiltration, bronchoalveolar lavage concentrations of CXC chemokines, and airway hyperreactivity. Neutralization of airway keratinocyte-derived chemokine and macrophage inflammatory protein-2 significantly improves the respiratory function in addition to decreasing the infiltration of neutrophils and eosinophils. Blocking the chemokines also decreased airway mucus production. These results demonstrate that DEP exacerbates airway inflammation induced by allergen through increased pulmonary expression of the CXC chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein-2).

Assessing Pulmonary Pathology by Detailed Examination of Respiratory Function

Pulmonary inflammation causes multiple alterations within the lung, including mucus production, recruitment of inflammatory cells, and airway hyperreactivity (AHR). Measurement of AHR by direct, invasive means (eg, mechanical ventilation) or noninvasive techniques, like whole body plethysmography (WBP), assesses the severity of pulmonary inflammation in animal models of inflammatory lung disease. Direct measurement of AHR is acknowledged as the most accurate method for assessing airway mechanics, but analysis of all data obtained from WBP may offer insights into which inflammatory aspects of the lung are altered along with AHR. Using WBP, we compared the respiratory parameters of two groups of mice sensitized with cockroach allergen. One group was treated with dexamethasone (Dex) before final challenge (Dex-Asthma), while the other group received vehicle treatment (Asthma). Respiratory parameters from plethysmography revealed that Dex-Asthma mice compensated to maintain high minute ventilation, whereas Asthma mice showed significant impairment in minute ventilation despite increased peak expiratory flow (103 ± 5 ml/min vs. 69 ± 70 ml/min). The WBP data suggest that enhanced air exchange in the Dex-Asthma mice results from significant decreases in airway mucus production. Additional studies with quantitative morphometry of histological sections confirmed that Dex reduced airway mucus. In conclusion, a detailed examination of WBP parameters can accurately assess the respiratory health of mice and will help direct additional studies.

Oral tolerance inhibits pulmonary eosinophilia in a cockroach allergen induced model of asthma: a randomized laboratory study.

BackgroundAntigen desensitization through oral tolerance is becoming an increasingly attractive treatment option for allergic diseases. However, the mechanism(s) by which tolerization is achieved remain poorly defined. In this study we endeavored to induce oral tolerance to cockroach allergen (CRA: a complex mixture of insect components) in order to ameliorate asthma-like, allergic pulmonary inflammation.MethodsWe compared the pulmonary inflammation of mice which had received four CRA feedings prior to intratracheal allergen sensitization and challenge to mice fed PBS on the same time course. Respiratory parameters were assessed by whole body unrestrained plethysmography and mechanical ventilation with forced oscillation. Bronchoalveolar lavage fluid (BAL) and lung homogenate (LH) were assessed for cytokines and chemokines by ELISA. BAL inflammatory cells were also collected and examined by light microscopy.ResultsCRA feeding prior to allergen sensitization and challenge led to a significant improvement in respiratory health. Airways hyperreactivity measured indirectly via enhanced pause (Penh) was meaningfully reduced in the CRA-fed mice compared to the PBS fed mice (2.3 ± 0.4 vs 3.9 ± 0.6; p = 0.03). Directly measured airways resistance confirmed this trend when comparing the CRA-fed to the PBS-fed animals (2.97 ± 0.98 vs 4.95 ± 1.41). This effect was not due to reduced traditional inflammatory cell chemotactic factors, Th2 or other cytokines and chemokines. The mechanism of improved respiratory health in the tolerized mice was due to significantly reduced eosinophil numbers in the bronchoalveolar lavage fluid (43300 ± 11445 vs 158786 ± 38908; p = 0.007) and eosinophil specific peroxidase activity in the lung homogenate (0.59 ± 0.13 vs 1.19 ± 0.19; p = 0.017). The decreased eosinophilia was likely the result of increased IL-10 in the lung homogenate of the tolerized mice (6320 ± 354 ng/mL vs 5190 ± 404 ng/mL, p = 0.02).ConclusionOur results show that oral tolerization to CRA can improve the respiratory health of experimental mice in a CRA-induced model of asthma-like pulmonary inflammation by reducing pulmonary eosinophilia.

Reducing LPS content in cockroach allergens increases pulmonary cytokine production without increasing inflammation: a randomized laboratory study.

BackgroundEndotoxins are ubiquitously present in the environment and constitute a significant component of ambient air. These substances have been shown to modulate the allergic response, however a consensus has yet to be reached whether they attenuate or exacerbate asthmatic responses. The current investigation examined whether reducing the concentration of lipopolysaccharide (LPS) in a house dust extract (HDE) containing high concentrations of both cockroach allergens [1] and LPS would attenuate asthma-like pulmonary inflammation.MethodsMice were sensitized with CRA and challenged with the intact HDE, containing 182 ng of LPS, or an LPS-reduced HDE containing 3 ng LPS, but an equivalent amount of CRA. Multiple parameters of asthma-like pulmonary inflammation were measured.ResultsCompared to HDE challenged mice, the LPS-reduced HDE challenged mice had significantly reduced TNFα levels in the bronchoalveolar lavage fluid. Plasma levels of IgE and IgG1 were significantly reduced, however no change in CRA-specific IgE was detected. In HDE mice, plasma IgG2a levels were similar to naïve mice, while LPS-reduced HDE mice had significantly greater concentrations. Reduced levels of LPS in the HDE did not decrease eosinophil or neutrophil recruitment into the alveolar space. Equivalent inflammatory cell recruitment occurred despite having generally higher pulmonary concentrations of eotaxins and CXC chemokines in the LPS-reduced HDE group. LPS-reduced HDE challenge induced significantly higher concentrations of IFNγ, and IL-5 and IL-13 in the BAL fluid, but did not decrease airways hyperresponsiveness or airway resistance to methacholine challenge. Conclusion: These data show that reduction of LPS levels in the HDE does not significantly protect against the severity of asthma-like pulmonary inflammation.

CHRONIC PULMONARY LPS TOLERANCE INDUCES SELECTIVE IMMUNOSUPPRESSION WHILE MAINTAINING THE NEUTROPHILIC RESPONSE

LPS challenge causes potent activation of innate immunity. Because LPS is ubiquitously present in ambient air, repeated inhalation may lead to activation of the pulmonary immune response. If this activation is unregulated, chronic LPS inhalation would lead to persistent inflammation and organ damage. We hypothesized that the lung uses the mechanism of LPS tolerance to maintain the balance between hypoinflammatory and hyperinflammatory states. We developed a model of chronic pulmonary LPS tolerance induced by pulmonary exposure to 1 &mgr;g LPS for 4 consecutive days. Mice were challenged with 10 &mgr;g of LPS 24 h later. TNF-&agr; protein was significantly decreased in the bronchoalveolar lavage fluid of tolerant versus nontolerant mice, whereas IL-6 levels were significantly increased in the tolerant group. Tolerant mice were also protected from airway hyperresponsiveness. M2 and M3 muscarinic receptor mRNA was significantly decreased in the lungs of tolerant mice, suggesting a mechanism for the decreased airway hyperresponsiveness. CXCL2 was significantly reduced in tolerant mice, but CXCL1 was equivalent between groups. No difference was seen in neutrophil recruitment to the alveolar space. Interestingly, LPS tolerance does not confer cross-tolerance to the Toll-like receptor (TLR) 2 stimulus Pam3Cys. TNF-&agr; and IL-6 concentrations were significantly increased in LPS-tolerant mice challenged with Pam3Cys; however, chemokine concentrations were unaffected. Our data show that repeated LPS inhalation results in differential regulation of cytokines but does not inhibit neutrophil recruitment. This unrestricted neutrophil recruitment may represent a mechanism by which individuals may be protected from pulmonary bacterial infection and pneumonia.

Pulmonary Endotoxin Tolerance Protects against Cockroach Allergen-Induced Asthma-Like Inflammation in a Mouse Model

Background: Compounds which activate the innate immune system, such as lipopolysaccharide, are significant components of ambient air, and extremely difficult to remove from the environment. It is currently unclear how prior inhalation of endotoxin affects allergen sensitization. We examined whether lung-specific endotoxin tolerance induction prior to sensitization can modulate the response to allergen. Methods: Endotoxin tolerance was induced by repeated intratracheal exposure to endotoxin. All mice were then sensitized and challenged by direct intratracheal instillation of cockroach allergen. Results: After allergen sensitization and challenge, endotoxin tolerant mice had significantly decreased airways hyperresponsiveness to methacholine challenge, which was confirmed by invasive lung function tests. Decreased goblet cell hyperplasia and mucus production were also found by histological assessment. Tolerant mice were protected from airway eosinophilia through the mechanism of reduced CCL11 and CCL24. Interestingly, endotoxin tolerant mice had only a modest reduction in cockroach-specific IgE; however, total IgE was significantly reduced. Conclusions: These data show that induction of endotoxin tolerance prior to sensitization protects against the hallmark features of asthma-like inflammation, and that transient modulation of innate immunity can have long-lasting effects on adaptive responses.

ELISA rescue protocol: Recovery of sample concentrations from an assay with an unsuccessful standard curve

The Enzyme-linked Immunosorbant Assay (ELISA) is a method commonly used to measure proteins in various biological matrices, due to its ease of performance and relatively low cost. In order for quantitative data to be generated, a reference standard curve must be prepared for each assay; however, due to investigator error or standard protein degradation, otherwise representative experimental sample data are rendered useless. Herein, we describe a protocol by which sample concentrations can be recovered from assays in which the standard curve fails. The ΔOD values of the experimental samples are used to generate a new standard curve, which is applied back to the original plate. For validation of this method, experimental sample concentrations obtained using acceptable standard curves were potted against those calculated using this new method. Using linear regression analysis, we show a near 1:1 correlation between sample concentrations, with r(2) values between 0.98 and 0.99 and slopes between 0.97 and 1.10. This method demonstrates that assays resulting in unusable standard curves do not require re-assay of all samples. Instead, the experimental sample concentrations can be retrieved saving the investigator the time and resources required to rerun samples or repeat entire experiments.

Herbal medicine treatment reduces inflammation in a murine model of cockroach allergen–induced asthma

BACKGROUND Asthma is a significant disease among children, and its prevalence has increased notably during the last 2 decades. A traditional Korean medicine, So-Cheong-Ryong-Tang (SCRT), has been used for the treatment of asthma in Asia for centuries, but its mechanism for reducing bronchopulmonary inflammation in asthma has yet to be elucidated. OBJECTIVE To investigate whether the herbal extract SCRT inhibits inflammation in a mouse model of cockroach allergen-induced asthma. METHODS A house dust extract containing endotoxin and cockroach allergens was used for immunization and 2 additional pulmonary challenges in BALB/c mice. Mice were treated with SCRT or vehicle 1 hour before each pulmonary challenge. Respiratory parameters were evaluated by whole-body plethysmography and forced oscillation methods 24 hours after the last challenge. Bronchoalveolar lavage (BAL) fluid was collected, and histologic sections of lung were prepared either 4 or 24 hours after the last house dust extract challenge. RESULTS SCRT treatment significantly reduced the hyperreactivity of the airways as measured by whole-body plethysmography and direct measurement of airway resistance. Inflammation was significantly inhibited by SCRT treatment as demonstrated by reduced plasma IgE levels and improved pulmonary histologic characteristics. SCRT significantly reduced the number of neutrophils in the BAL fluid and also significantly reduced the BAL levels of CXC chemokines, providing a potential mechanism for the reduced inflammation. In a similar fashion, SCRT reduced eosinophil recruitment and BAL levels of eotaxin and RANTES. CONCLUSION These data indicate that SCRT treatment alleviates asthma-like pulmonary inflammation via suppression of specific chemokines.