Surfactant Protein A Enhances the Degradation of LPS-Induced TLR4 in Primary Alveolar Macrophages Involving Rab7, ß-arrestin2, and mTORC1.

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A-/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNF-α release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires ß-arrestin2 and, in ß-arrestin2-/- AMs and after intratracheal LPS challenge of ß-arrestin2-/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A-/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires ß-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, ß-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.

Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung.

Dose escalation for metastatic spinal cord compression in patients with relatively radioresistant tumors.

PURPOSE: Radiotherapy alone is the most common treatment for metastatic spinal cord compression (MSCC) from relatively radioresistant tumors such as renal cell carcinoma, colorectal cancer, and malignant melanoma. However, the results of the "standard" regimen 30 Gy/10 fractions need to be improved with respect to functional outcome. This study investigated whether a dose escalation beyond 30 Gy can improve treatment outcomes. METHODS AND MATERIALS: A total of 91 patients receiving 30 Gy/10 fractions were retrospectively compared to 115 patients receiving higher doses (37.5 Gy/15 fractions, 40 Gy/20 fractions) for motor function and local control of MSCC. Ten further potential prognostic factors were evaluated: age, gender, tumor type, performance status, number of involved vertebrae, visceral or other bone metastases, interval from tumor diagnosis to radiotherapy, pretreatment ambulatory status, and time developing motor deficits before radiotherapy. RESULTS: Motor function improved in 18% of patients after 30 Gy and in 22% after higher doses (p = 0.81). On multivariate analysis, functional outcome was associated with visceral metastases (p = 0.030), interval from tumor diagnosis to radiotherapy (p = 0.010), and time developing motor deficits (p < 0.001). The 1-year local control rates were 76% after 30 Gy and 80% after higher doses, respectively (p = 0.64). On multivariate analysis, local control was significantly associated with visceral metastases (p = 0.029) and number of involved vertebrae (p = 0.043). CONCLUSIONS: Given the limitations of a retrospective study, escalation of the radiation dose beyond 30 Gy/10 fractions did not significantly improve motor function and local control of MSCC in patients with relatively radioresistant tumors.

Radiotherapy for oligometastatic disease in patients with spinal cord compression (MSCC) from relatively radioresistant tumors.

BACKGROUND: Radiotherapy alone is the most common treatment for metastatic spinal cord compression (MSCC). Patients with relatively radioresistant tumors and oligometastatic disease may benefit from more intensive therapies (surgery, high-precision radiotherapy). If such therapies are not available, one can speculate whether patients benefit from dose escalation beyond the standard regimen 30 Gy in ten fractions. PATIENTS AND METHODS: Of 206 patients with MSCC from relatively radioresistant tumors (renal cell carcinoma, colorectal cancer, malignant melanoma), 51 had oligometastatic disease (no visceral or other bone metastases, involvement of only one to three vertebrae). In this subset, 21 patients receiving 30 Gy in ten fractions were retrospectively compared to 30 patients receiving higher doses. Seven further potential prognostic factors were investigated: age, gender, tumor type, performance status, interval from tumor diagnosis to radiotherapy of MSCC, pretreatment ambulatory status, and time developing motor deficits before radiotherapy. RESULTS: Motor function improved in 52% of patients after 30 Gy and 40% after higher doses (p = 0.44). On multivariate analysis, functional outcome was associated with interval from tumor diagnosis to radiotherapy (p = 0.020). 1-year local control rates were 84% after 30 Gy and 82% after higher doses (p = 0.75). No factor was associated with local control. 1-year survival rates were 76% after 30 Gy and 63% after higher doses (p = 0.52). On multivariate analysis, survival was associated with performance status (p = 0.022) and interval from tumor diagnosis to radiotherapy (p = 0.039), and almost with pretreatment ambulatory status (p = 0.069). CONCLUSION: Dose escalation beyond 30 Gy in ten fractions did not improve motor function, local control, and survival in MSCC patients with oligometastatic disease from relatively radioresistant tumors.