Context-dependent roles for autophagy in myeloid cells in tumor progression.

Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy-deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.

Number of sinus procedures reduces inpatient mortality for invasive fungal rhinosinusitis.

OBJECTIVES: To evaluate surgical outcomes of invasive fungal rhinosinusitis MATERIALS AND METHODS: The National Inpatient Sample Database (2000-2015 Q3) was queried for patients with a diagnosis of aspergillosis and/or mucormycosis and a diagnosis of acute sinusitis using the International Classification of Diseases, Ninth Edition. Factors associated with inpatient mortality were then identified with multivariate logistic regression. RESULTS: 514 adult patients with a median age of 57.0 years were identified, of which 231 (44.9 %) underwent sinus surgery. Surgical patients had a longer length of stay (17.0 vs 9.0 days, p < 0.001) and higher total charges ($139,762.00 vs $57,945.00, p < 0.001). The number of sinus procedures was associated with reduced odds of inpatient mortality (OR 0.69; p < 0.001) in multivariate analysis. Hypertension (OR 0.34, p = 0.002) and chronic kidney disease (OR 0.23, p = 0.034) were associated with reduced odds of inpatient mortality. Total number of procedures (OR 1.24; p = 0.002), mucormycosis (OR 2.75, p = 0.002), age (OR 1.03, p = 0.006) and acid-base disorders (OR 2.85, p = 0.012) were associated with increased odds of inpatient mortality. CONCLUSION: This represents the first large scale study to evaluate outcomes for invasive fungal rhinosinusitis. These findings suggest the odds of inpatient mortality decrease with greater extent of sinus surgery performed. The potentially protective roles of hypertension and chronic kidney disease should be evaluated in future research.

Patient Satisfaction with Nonopioid Postoperative Analgesia in Head and Neck Surgery: A Prospective Randomized Trial.

OBJECTIVE: To evaluate patients' satisfaction with opioid versus opioid-sparing postoperative analgesia in patients undergoing outpatient head and neck surgery. STUDY DESIGN: Prospective randomized trial. SETTING: Tertiary care academic hospital. METHODS: Adult patients undergoing outpatient head and neck surgery were randomly assigned to 1 of 3 analgesic regimens. First- and second-line medications were the following by group (1) Hydrocodone-acetaminophen with ibuprofen, (2) ibuprofen with hydrocodone-acetaminophen, and (3) ibuprofen with acetaminophen. Preoperative counseling was provided to patients regarding expected pain and proper medication use. Postoperative questionnaires were administered to assess satisfaction. RESULTS: One hundred three patients were enrolled in the study (mean age, 56.5 years; women, 75 [73%]). The mean satisfaction score with the pain regimen assigned was similar between the 3 groups (scale 0-10, [7.7, 8.3, 8.5, P = .46]). A similar percentage of patients in each group reported that surgery was more painful than anticipated (25%, 32%, 26%, P = .978), and a similar percentage of patients reported willingness to utilize the same analgesic regimen following future surgeries (75%, 83%, 76%, P = .682). Additional questions evaluating the side effect profile, maximum and minimum pain scores, and difficulty of recovery were not statistically different between the 3 groups. CONCLUSION: In the postoperative population for outpatient head and neck surgeries, there was no significant difference in patient satisfaction and pain control between the opioid and nonopioid arms. Providers should discuss opioid-sparing regimens preoperatively with patients and describe them as effective in providing adequate pain control without a significant impact on patient's perception of care.

Nomenclature of the radial nerve: distinguishing between the deep branch of the radial nerve and the posterior interosseous nerve.

To address an inconsistency in the nomenclature of the anatomy and compressive syndromes of the deep branch of the radial nerve, we advocate for a single compression syndrome that presents along a spectrum from pain to posterior interosseous nerve palsy.

Artificial intelligence-based epigenomic, transcriptomic and histologic signatures of tobacco use in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) biomarker studies rarely employ multi-omic biomarker strategies and pertinent clinicopathologic characteristics to predict mortality. In this study we determine for the first time a combined epigenetic, gene expression, and histology signature that differentiates between patients with different tobacco use history (heavy tobacco use with ≥10 pack years vs. no tobacco use). Using The Cancer Genome Atlas (TCGA) cohort (n = 257) and an internal cohort (n = 40), we identify 3 epigenetic markers (GPR15, GNG12, GDNF) and 13 expression markers (IGHA2, SCG5, RPL3L, NTRK1, CD96, BMP6, TFPI2, EFEMP2, RYR3, DMTN, GPD2, BAALC, and FMO3), which are dysregulated in OSCC patients who were never smokers vs. those who have a ≥ 10 pack year history. While mortality risk prediction based on smoking status and clinicopathologic covariates alone is inaccurate (c-statistic = 0.57), the combined epigenetic/expression and histologic signature has a c-statistic = 0.9409 in predicting 5-year mortality in OSCC patients.

Correlative multiscale 3D imaging of mouse primary and metastatic tumors by sequential light sheet and confocal fluorescence microscopy.

Three-dimensional (3D) optical microscopy, combined with advanced tissue clearing, permits in situ interrogation of the tumor microenvironment (TME) in large volumetric tumors for preclinical cancer research. Light sheet (also known as ultramicroscopy) and confocal fluorescence microscopy are often used to achieve macroscopic and microscopic 3D images of optically cleared tumor tissues, respectively. Although each technique offers distinct fields of view (FOVs) and spatial resolution, the combination of these two optical microscopy techniques to obtain correlative multiscale 3D images from the same tumor tissues has not yet been explored. To establish correlative multiscale 3D optical microscopy, we developed a method for optically marking defined regions of interest (ROIs) within a cleared mouse tumor by employing a UV light-activated visible dye and Z-axis position-selective UV irradiation in a light sheet microscope system. By integrating this method with subsequent tissue processing, including physical ROI marking, reversal of tissue clearing, tissue macrosectioning, and multiplex immunofluorescence, we established a workflow that enables the tracking and 3D imaging of ROIs within tumor tissues through sequential light sheet and confocal fluorescence microscopy. This approach allowed for quantitative 3D spatial analysis of the immune response in the TME of a mouse mammary tumor following cancer immunotherapy at multiple spatial scales. The workflow also facilitated the direct localization of a metastatic lesion within a whole mouse brain. These results demonstrate that our ROI tracking method and its associated workflow offer a novel approach for correlative multiscale 3D optical microscopy, with the potential to provide new insights into tumor heterogeneity, metastasis, and response to therapy at various spatial levels.

Increased Multiplexity in Optical Tissue Clearing-Based Three-Dimensional Immunofluorescence Microscopy of the Tumor Microenvironment by Light-Emitting Diode Photobleaching.

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 µm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.

Anatomical front and back reconstruction sustains radiographic alignment: a comparison to three-ligament tenodesis.

Multiple techniques exist to reconstruct the scapholunate interosseous ligament, though none have demonstrated superiority. This study compares 1-year radiographic outcomes of the three-ligament tenodesis and the anatomical front and back reconstruction. All patients who underwent reconstruction of their scapholunate interosseous ligament at one institution with either anatomical front and back reconstruction or three-ligament tenodesis between 2011 and 2020 were retrospectively reviewed. At 52-week follow-up, anatomical front and back reconstruction maintained a statistically significant improvement in scapholunate gap, corrected radiolunate angle and dorsal scaphoid translation, while three-ligament tenodesis demonstrated no sustained improvement in any parameter. The improvement in dorsal scaphoid translation was significantly greater for patients undergoing anatomical front and back reconstruction compared with three-ligament tenodesis at the 16-weeks postoperative timepoint (-1.0 mm, -0.3 mm). Anatomical front and back reconstruction demonstrates sustained improvement in radiographic outcomes at 1 year when compared with three-ligament tenodesis. By addressing both volar and dorsal critical ligament restraints, adoption of anatomical front and back reconstruction for advanced stage scapholunate interosseous ligament injuries should be considered.Level of evidence: IV.

Increased multiplexity in optical tissue clearing-based 3D immunofluorescence microscopy of the tumor microenvironment by LED photobleaching.

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy have been transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only three or four cellular and non-cellular TME components can be localized in a cleared tumor tissue. Here we report a LED photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through three work cycles, we produced 8-plex image data from individual 400 µm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.

Factors Influencing Surgical Decision Making in Trapeziometacarpal Osteoarthritis.

Purpose: With trapeziometacarpal osteoarthritis (TMC OA), the relationship between disease severity and pretreatment dysfunction, patient expectations, and preferred patient treatment and management remains unclear. This study aimed to assess the association between functional status, pretreatment expectations, and demographic and clinical characteristics of TMC OA patients who decide to undergo operative management. Methods: Patients diagnosed with TMC OA (n = 96) were administered the Thumb Arthritis Expectations Survey and the Brief Michigan Hand Questionnaire (bMHQ) during their initial office visit. Demographic data (sex, age, race, education level, marital status, comorbidities, and hand dominance) and clinical characteristics (prior injury, and therapeutic interventions including splinting, steroid injections, therapy, and anti-inflammatory medication) were collected. Multiple logistic regression was used to assess the association between surgical treatment and expectation scores. Results: Our logistic regression model found that lower bMHQ scores, high thumb arthritis expectation survey scores, and prior treatments for TMC OA were associated significantly with the surgical treatment of TMC OA. After controlling for all possible covariates, the odds of having surgery was 3.9 times higher among patients with high expectations (above median) compared to patients with low expectations (adjusted odds ratio [AOR], 3.9; 95% confidence interval [CI], 1.3-11.2). Patients with average function, as measured by bMHQ scores, were 74.5% less likely to elect for surgery than those with the lowest bMHQ (AOR, 0.3; 95% CI, 0.1-0.9). Patients treated previously with steroids were 13 times more likely to elect for surgery than those who were never treated for TMC arthritis (AOR,13.1; 95% CI, 2.2-77.0). Conclusions: Patients with TMC OA who elect to proceed with surgical management have lower bMHQ (greater perceived dysfunction) and higher expectations, and have had prior treatment. Age was not a significant predictor of surgical management of TMC OA. Type of study/level of evidence: Prognostic IV.

Development and Validation of a Survey to Measure Patients' Expectations of Wrist Arthritis Surgery.

Background The purpose of this study was to develop and test a patient-derived expectations survey for wrist arthritis surgery. We hypothesized that preoperative patient expectations are higher in people with greater functional impairment and that postoperative fulfilment of patient expectations correlates with functional improvement. Methods The study was conducted in four phases. Development (n = 22) Preoperatively, patients were asked open-ended questions regarding expectations of surgery. A draft survey was then assembled. Reliability (n = 35) The survey was administered twice preoperatively. Concordance was measured with weighted kappa values and intraclass correlations (ICC). Validity (n = 58) Validity was assessed by comparing responses from the Expectations Survey to the patient-rated wrist evaluation (PRWE). Responsiveness (n = 18) Responsiveness was calculated by comparing the proportion of expectations fulfilled to PRWE scores 1-year postoperatively. Results Development Twenty-two distinct items representing the most frequent responses were utilized from the draft survey items of 1,244 expectations volunteered. Reliability Patients had high preoperative expectations of surgery (mean = 76.8); 30% had scores ≥90. Test-retest reliability was high (Cronbach α coefficients = 0.91, 0.93, ICC = 0.86). Endorsement of items = 66 to 100%; and weighted kappa values = 0.39 to 0.96. Validity Patients with greater preoperative expectations (≥63) had more pain, worse function, and worse PRWE scores than those with lower expectations. Responsiveness The proportion of fulfilled expectations was high (mean 0.80, median 0.79), and greater fulfillment (proportion > 0.80, n = 8) was associated with better postoperative PRWE scores. Conclusion The patient-derived expectations survey is reliable, valid, responsive, and addresses a spectrum of expectations for patients undergoing surgery for wrist arthritis. Clinical relevance Understanding patient expectations can contribute to customized care given the range of surgical choices for the arthritic wrist.

Biomechanical analysis of three techniques of suspensionplasty after trapeziectomy: a cadaveric study.

The aim of the present cadaveric study was to assess resistance to first metacarpal subsidence of three techniques of suspensionplasty after trapeziectomy. In total, 18 forearms (mean age 60 years [range 20-89]) were used with six specimens per surgical technique: palmar oblique ligament reconstruction with tendon interposition (LRTI), abductor pollicis longus (APL) suspensionplasty, or suture suspensionplasty. There was no significant difference in mean trapezial space height after trapeziectomy and suspensionplasty compared to the preoperative trapezial height. However, after simulation of physiological lateral pinch, there was a significant (p < 0.05) difference in mean trapezial space height between the APL suspensionplasty and the suture suspensionplasty compared to the LRTI group. After axial loading, there was significantly greater metacarpal subsidence in the LRTI group compared to the APL and suture suspensionplasty groups but no statistically significant difference between the suture suspensionplasty and the APL suspensionplasty groups.Level of evidence: V.

Transoral Laser Resection of the Tongue Base in the Workup of Unknown Primary Head and Neck Squamous Cell Carcinoma.

Objective Failure to localize the primary tumor site in head and neck carcinoma of unknown origin after imaging and endoscopic evaluation leads to increased treatment-related morbidity. The use of transoral laser microsurgery to improve the detection of unknown primary carcinoma site identification is described in this article. Methods A retrospective cohort of 71 consecutive cases of cervical carcinoma of an unknown primary source from 2006 until 2012 from a single academic institution was analyzed. Of these, 10 patients were excluded based on our exclusion criteria. All patients underwent endoscopy with biopsies performed by fellowship-trained head and neck cancer surgeons.  Results The primary detection rate was 76% for patients who underwent laser tongue base resection versus 34% for traditional operative examination. There were no complications or prolonged recovery times in either group. Operative time was increased by the addition of the transoral base of tongue resection by 30 minutes. Conclusions Laser tongue base excision offers improved sensitivity in primary site detection without a significant increase in morbidity and only a modest increase in operative time.

The Influence of Psychosocial Factors on Disability and Expected Improvement Before Surgery for Adult Traumatic Brachial Plexus Injury.

PURPOSE: Psychosocial factors influence pain and recovery after extremity trauma and may be targets for early intervention. This may be of particular interest for patients with adult traumatic brachial plexus injury (BPI), given the broad and devastating impact of the injury. We hypothesized that there would be an association between depressive symptoms, anxiety, and pain interference with preoperative disability and expectations for improvement after BPI surgery. METHODS: We enrolled 34 patients into a prospective multicenter cohort study for those undergoing surgery for adult traumatic BPI. Before surgery, participants completed Patient-Reported Outcome Measurement Information System scales for pain interference, anxiety, and depressive symptoms, and a validated BPI-specific measure of disability and expected improvement. We performed Pearson correlation analysis between pain interference, anxiety symptoms, and depressive symptoms with (A) disability and (B) expected improvement. We created separate linear regression models for (A) disability and (B) expected improvement including adjustment for severity of plexus injury, age, sex, and race. RESULTS: Among 34 patients, there was a moderate, statistically significant, correlation between preoperative depressive symptoms and higher disability. This remained significant in a linear regression model adjusted for severity of plexus injury, age, sex, and race. There was no association between severity of plexus injury and disability. Depressive symptoms also were moderately, but significantly, correlated with higher expected improvement. This remained significant in a linear regression model adjusted for severity of plexus injury, age, sex, and race. CONCLUSIONS: Depressive symptoms are associated with greater disability and higher expected improvement before BPI surgery. Screening for depressive symptoms can help BPI teams identify patients who would benefit from early referral to mental health specialists and tailor appropriate expectations counseling for functional recovery. We did not find an association between severity of BPI and patient-reported disability, suggesting either that the scale may lack validity or that the sample is biased. LEVEL OF EVIDENCE: Prognostic II.

Referral Patterns Associated With Delayed Care for Traumatic Brachial Plexus Injuries.

PURPOSE: Traumatic brachial plexus injuries (BPI) are devastating, time-sensitive conditions that often require definitive treatment at academic tertiary care centers. Delays to presentation and surgery have been associated with inferior outcomes. In this study, we evaluate referral patterns associated with delayed presentation and late surgery in traumatic BPI patients. METHODS: Patients diagnosed with a traumatic BPI at our institution from 2000 to 2020 were identified. Medical charts were reviewed for demographic characteristics, prereferral work-up, and referring provider characteristics. Delayed presentation was defined as greater than 3 months from date of injury to initial evaluation by our brachial plexus specialists. Late surgery was defined as greater than 6 months from date of injury. Multivariable logistic regression was used to identify factors associated with delayed presentation or surgery. RESULTS: A total of 99 patients were included, 71 of whom underwent surgery. Sixty-two patients presented delayed (62.6%), with 26 receiving late surgery (36.6%). There were similar rates of delayed presentation or late surgery by referring provider specialty. Patients whose initial diagnostic EMG was ordered by the referring provider prior to initial presentation at our institution were more likely to have a delayed presentation (76.2% vs 31.3%) and undergo late surgery (44.9% vs 10.0%). CONCLUSIONS: Delayed presentation and late surgery in traumatic BPI patients were associated with initial diagnostic EMG ordered by the referring provider. CLINICAL RELEVANCE: Delayed presentation and surgery have been associated with inferior outcomes in traumatic BPI patients. We recommend that providers direct patients with clinical concern of traumatic BPI directly to a brachial plexus center without further work-up before referral and encourage referral centers to accept these patients.

Targeting Tumor Microenvironment Akt Signaling Represents a Potential Therapeutic Strategy for Aggressive Thyroid Cancer.

Effects of the tumor microenvironment (TME) stromal cells on progression in thyroid cancer are largely unexplored. Elucidating the effects and underlying mechanisms may facilitate the development of targeting therapy for aggressive cases of this disease. In this study, we investigated the impact of TME stromal cells on cancer stem-like cells (CSCs) in patient-relevant contexts where applying in vitro assays and xenograft models uncovered contributions of TME stromal cells to thyroid cancer progression. We found that TME stromal cells can enhance CSC self-renewal and invasiveness mainly via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. The disruption of Akt signaling could diminish the impact of TME stromal cells on CSC aggressiveness in vitro and reduce CSC tumorigenesis and metastasis in xenografts. Notably, disrupting Akt signaling did not cause detectable alterations in tumor histology and gene expression of major stromal components while it produced therapeutic benefits. In addition, using a clinical cohort, we discovered that papillary thyroid carcinomas with lymph node metastasis are more likely to have elevated Akt signaling compared with the ones without metastasis, suggesting the relevance of Akt-targeting. Overall, our results identify PI3K/Akt pathway-engaged contributions of TME stromal cells to thyroid tumor disease progression, illuminating TME Akt signaling as a therapeutic target in aggressive thyroid cancer.

GA-OH enhances the cytotoxicity of photon and proton radiation in HPV+ HNSCC cells.

Introduction: Treatment-related toxicity following either chemo- or radiotherapy can create significant clinical challenges for HNSCC cancer patients, particularly those with HPV-associated oropharyngeal squamous cell carcinoma. Identifying and characterizing targeted therapy agents that enhance the efficacy of radiation is a reasonable approach for developing de-escalated radiation regimens that result in less radiation-induced sequelae. We evaluated the ability of our recently discovered, novel HPV E6 inhibitor (GA-OH) to radio-sensitize HPV+ and HPV- HNSCC cell lines to photon and proton radiation. Methods: Radiosensitivity to either photon or proton beams was assessed using various assays such as colony formation assay, DNA damage markers, cell cycle and apoptosis, western blotting, and primary cells. Calculations for radiosensitivity indices and relative biological effectiveness (RBE) were based on the linear quadratic model. Results: Our results showed that radiation derived from both X-ray photons and protons is effective in inhibiting colony formation in HNSCC cells, and that GA-OH potentiated radiosensitivity of the cells. This effect was stronger in HPV+ cells as compared to their HPV- counterparts. We also found that GA-OH was more effective than cetuximab but less effective than cisplatin (CDDP) in enhancing radiosensitivity of HSNCC cells. Further tests indicated that the effects of GA-OH on the response to radiation may be mediated through cell cycle arrest, particularly in HPV+ cell lines. Importantly, the results also showed that GA-OH increases the apoptotic induction of radiation as measured by several apoptotic markers, even though radiation alone had little effect on apoptosis. Conclusion: The enhanced combinatorial cytotoxicity found in this study indicates the strong potential of E6 inhibition as a strategy to sensitize cells to radiation. Future research is warranted to further characterize the interaction of GA-OH derivatives and other E6-specific inhibitors with radiation, as well as its potential to improve the safety and effectiveness of radiation treatment for patients with oropharyngeal cancer.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Spatially selective cell treatment and collection for integrative drug testing using hydrodynamic flow focusing and shifting.

Hydrodynamic focusing capable of readily producing and controlling laminar flow facilitates drug treatment of cells in existing microfluidic culture devices. However, to expand applications of such devices to multiparameter drug testing, critical limitations in current hydrodynamic focusing microfluidics must be addressed. Here we describe hydrodynamic focusing and shifting as an advanced microfluidics tool for spatially selective drug delivery and integrative cell-based drug testing. We designed and fabricated a co-flow focusing, three-channel microfluidic device with a wide cell culture chamber. By controlling inlet flow rates of sample and two side solutions, we could generate hydrodynamic focusing and shifting that mediated precise regulation of the path and width of reagent and drug stream in the microfluidic device. We successfully validated a hydrodynamic focusing and shifting approach for spatially selective delivery of DiI, a lipophilic fluorophore, and doxorubicin, a chemotherapeutic agent, to tumor cells in our device. Moreover, subsequent flowing of a trypsin EDTA solution over the cells that were exposed to doxorubicin flow allowed us to selectively collect the treated cells. Our approach enabled downstream high-resolution microscopy of the cell suspension to confirm the nuclear delivery of doxorubicin into the tumor cells. In the device, we could also evaluate in situ the cytotoxic effect of doxorubicin to the tumor cells that were selectively treated by hydrodynamic flow focusing and shifting. These results show that hydrodynamic focusing and shifting enable a fast and robust approach to spatially treat and then collect cells in an optimized microfluidic device, offering an integrative assay tool for efficient drug screening and discovery.