Comparative effects of CFTR modulators on phagocytic, metabolic and inflammatory profiles of CF and nonCF macrophages.

Macrophage dysfunction has been well-described in Cystic Fibrosis (CF) and may contribute to bacterial persistence in the lung. Whether CF macrophage dysfunction is related directly to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in macrophages or an indirect consequence of chronic inflammation and mucostasis is a subject of ongoing debate. CFTR modulators that restore CFTR function in epithelial cells improve global CF monocyte inflammatory responses but their direct effects on macrophages are less well understood. To address this knowledge gap, we measured phagocytosis, metabolism, and cytokine expression in response to a classical CF pathogen, Pseudomonas aeruginosa in monocyte-derived macrophages (MDM) isolated from CF F508del homozygous subjects and nonCF controls. Unexpectedly, we found that CFTR modulators enhanced phagocytosis in both CF and nonCF cohorts. CFTR triple modulators also inhibited MDM mitochondrial function, consistent with MDM activation. In contrast to studies in humans where CFTR modulators decreased serum inflammatory cytokine levels, modulators did not alter cytokine secretion in our system. Our studies therefore suggest modulator induced metabolic effects may promote bacterial clearance in both CF and nonCF monocyte-derived macrophages.

The Impact of Chronic Electronic Cigarette Use on Alveolar Macrophage Lipid Content: Case Report.

Background: While acute respiratory distress following electronic cigarette (e-cig) use has been described, the effects of chronic e-cig use on lung health are currently unknown. Acute e-cigarette/vaping product use-associated lung injury (EVALI) has been highlighted recently in numerous cases across the United States. Numerous EVALI case reports highlight alterations in alveolar macrophages, justifying investigation of this key immune sentinel of the lung in habitual e-cig users. Case Presentation: After informed consent, we performed a bronchoscopy on a 25 year asymptomatic woman who reported daily e-cig use. To evaluate for evidence of abnormal lipid homeostasis, we performed histologic and Oil Red O stain evaluation of alveolar macrophages obtained from bronchoalveolar lavage fluid. Our analyses demonstrate a prevalence of cells with high lipid accumulation in multiple, discrete cytoplasmic foci. We found a high lipid laden macrophage index within alveolar macrophages isolated from a chronic e-cig user. At the ultrastructural level, we found membrane-bound compartments filled with material of various densities segregated along curved phase separation lines reminiscent of suspensions of immiscible fluids. Conclusions: We found a unique ultrastructural pattern in alveolar macrophages isolated from a chronic e-cig user that is unlike any other previously reported in aspiration syndromes and may represent a defining diagnostic feature of chronic e-cig use.

ScRNA-seq expression of IFI27 and APOC2 identifies four alveolar macrophage superclusters in healthy BALF.

Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs, interstitial macrophages, and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified two MARCKS+LGMN+IMs, FOLR2+SELENOP+ and SPP1+PLA2G7+ IMs, monocyte subtypes, DC1, DC2, migDCs, plasmacytoid DCs, lymphocytes, epithelial cells, and four AM superclusters (families) based on the gene expression of IFI27 and APOC2 These four AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, cholesterol, chemokine, metallothionein, interferon, and small-cluster AMs. Interestingly, the chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Last, other AM datasets were projected onto our dataset. Similar AM superclusters and functional subclusters were observed, along with a significant increase in chemokine and IFN AM subclusters in individuals infected with COVID-19. Overall, functional specializations of the AM subclusters suggest that there are highly regulated AM niches with defined programming states, highlighting a clear division of labor.

Exposure to extracellular vesicles from Pseudomonas aeruginosa result in loss of DNA methylation at enhancer and DNase hypersensitive site regions in lung macrophages.

Various pathogens use differing strategies to evade host immune response including modulating the host's epigenome. Here, we investigate if EVs secreted from P. aeruginosa alter DNA methylation in human lung macrophages, thereby potentially contributing to a dysfunctional innate immune response. Using a genome-wide DNA methylation approach, we demonstrate that P. aeruginosa EVs alter certain host cell DNA methylation patterns. We identified 1,185 differentially methylated CpGs (FDR < 0.05), which were significantly enriched for distal DNA regulatory elements including enhancer regions and DNase hypersensitive sites. Notably, all but one of the 1,185 differentially methylated CpGs were hypomethylated in association with EV exposure. Significantly hypomethylated CpGs tracked to genes including AXL, CFB and CCL23. Gene expression analysis identified 310 genes exhibiting significantly altered expression 48 hours post P. aeruginosa EV treatment, with 75 different genes upregulated and 235 genes downregulated. Some CpGs associated with cytokines such as CSF3 displayed strong negative correlations between DNA methylation and gene expression. Our infection model illustrates how secreted products (EVs) from bacteria can alter DNA methylation of the host epigenome. Changes in DNA methylation in distal DNA regulatory regions in turn can modulate cellular gene expression and potential downstream cellular processes.

Extracellular Vesicles from Pseudomonas aeruginosa Suppress MHC-Related Molecules in Human Lung Macrophages.

Pseudomonas aeruginosa, a Gram-negative bacterium, is one of the most common pathogens colonizing the lungs of cystic fibrosis patients. P. aeruginosa secrete extracellular vesicles (EVs) that contain LPS and other virulence factors that modulate the host's innate immune response, leading to an increased local proinflammatory response and reduced pathogen clearance, resulting in chronic infection and ultimately poor patient outcomes. Lung macrophages are the first line of defense in the airway innate immune response to pathogens. Proper host response to bacterial infection requires communication between APC and T cells, ultimately leading to pathogen clearance. In this study, we investigate whether EVs secreted from P. aeruginosa alter MHC Ag expression in lung macrophages, thereby potentially contributing to decreased pathogen clearance. Primary lung macrophages from human subjects were collected via bronchoalveolar lavage and exposed to EVs isolated from P. aeruginosa in vitro. Gene expression was measured with the NanoString nCounter gene expression assay. DNA methylation was measured with the EPIC array platform to assess changes in methylation. P. aeruginosa EVs suppress the expression of 11 different MHC-associated molecules in lung macrophages. Additionally, we show reduced DNA methylation in a regulatory region of gene complement factor B (CFB) as the possible driving mechanism of widespread MHC gene suppression. Our results demonstrate MHC molecule downregulation by P. aeruginosa-derived EVs in lung macrophages, which is consistent with an immune evasion strategy employed by a prokaryote in a host-pathogen interaction, potentially leading to decreased pulmonary bacterial clearance.

Functional and metabolic impairment in cigarette smoke-exposed macrophages is tied to oxidative stress.

Cigarette smoke inhalation exposes the respiratory system to thousands of potentially toxic substances and causes chronic obstructive pulmonary disease (COPD). COPD is characterized by cycles of inflammation and infection with a dysregulated immune response contributing to disease progression. While smoking cessation can slow the damage in COPD, lung immunity remains impaired. Alveolar macrophages (AMΦ) are innate immune cells strategically poised at the interface between lungs, respiratory pathogens, and environmental toxins including cigarette smoke. We studied the effects of cigarette smoke on model THP-1 and peripheral blood monocyte derived macrophages, and discovered a marked inhibition of bacterial phagocytosis which was replicated in primary human AMΦ. Cigarette smoke decreased AMΦ cystic fibrosis transmembrane conductance regulator (CFTR) expression, previously shown to be integral to phagocytosis. In contrast to cystic fibrosis macrophages, smoke-exposed THP-1 and AMΦ failed to augment phagocytosis in the presence of CFTR modulators. Cigarette smoke also inhibited THP-1 and AMΦ mitochondrial respiration while inducing glycolysis and reactive oxygen species. These effects were mitigated by the free radical scavenger N-acetylcysteine, which also reverted phagocytosis to baseline levels. Collectively these results implicate metabolic dysfunction as a key factor in the toxicity of cigarette smoke to AMΦ, and illuminate avenues of potential intervention.

DNA Methylation Changes in Regional Lung Macrophages Are Associated with Metabolic Differences.

A number of pulmonary diseases occur with upper lobe predominance, including cystic fibrosis and smoking-related chronic obstructive pulmonary disease. In the healthy lung, several physiologic and metabolic factors exhibit disparity when comparing the upper lobe of the lung to lower lobe, including differences in oxygenation, ventilation, lymphatic flow, pH, and blood flow. In this study, we asked whether these regional differences in the lung are associated with DNA methylation changes in lung macrophages that could potentially lead to altered cell responsiveness upon subsequent environmental challenge. All analyses were performed using primary lung macrophages collected via bronchoalveolar lavage from healthy human subjects with normal pulmonary function. Epigenome-wide DNA methylation was examined via Infinium MethylationEPIC (850K) array and validated by targeted next-generation bisulfite sequencing. We observed 95 CpG loci with significant differential methylation in lung macrophages, comparing upper lobe to lower lobe (all false discovery rate < 0.05). Several of these genes, including CLIP4, HSH2D, NR4A1, SNX10, and TYK2, have been implicated as participants in inflammatory/immune-related biological processes. Functionally, we identified phenotypic differences in oxygen use, comparing upper versus lower lung macrophages. Our results support a hypothesis that epigenetic changes, specifically DNA methylation, at a multitude of gene loci in lung macrophages are associated with metabolic differences regionally in lung.

Genome-wide DNA methylation profiling shows a distinct epigenetic signature associated with lung macrophages in cystic fibrosis.

BACKGROUND: Lung macrophages are major participants in the pulmonary innate immune response. In the cystic fibrosis (CF) lung, the inability of lung macrophages to successfully regulate the exaggerated inflammatory response suggests dysfunctional innate immune cell function. In this study, we aim to gain insight into innate immune cell dysfunction in CF by investigating alterations in DNA methylation in bronchoalveolar lavage (BAL) cells, composed primarily of lung macrophages of CF subjects compared with healthy controls. All analyses were performed using primary alveolar macrophages from human subjects collected via bronchoalveolar lavage. Epigenome-wide DNA methylation was examined via Illumina MethylationEPIC (850 K) array. Targeted next-generation bisulfite sequencing was used to validate selected differentially methylated CpGs. Methylation-based sample classification was performed using the recursively partitioned mixture model (RPMM) and was tested against sample case-control status. Differentially methylated loci were identified by fitting linear models with adjustment of age, sex, estimated cell type proportions, and repeat measurement. RESULTS: RPMM class membership was significantly associated with the CF disease status (P = 0.026). One hundred nine CpG loci were differentially methylated in CF BAL cells (all FDR ≤ 0.1). The majority of differentially methylated loci in CF were hypo-methylated and found within non-promoter CpG islands as well as in putative enhancer regions and DNase hyper-sensitive regions. CONCLUSIONS: These results support a hypothesis that epigenetic changes, specifically DNA methylation at a multitude of gene loci in lung macrophages, may participate, at least in part, in driving dysfunctional innate immune cells in the CF lung.

The mitogen-activated protein kinase pathway plays a critical role in regulating immunological properties of BRAF mutant cutaneous melanoma cells.

The advent of drugs targeting the mitogen-activated protein kinase (MAPK) pathway has markedly changed the treatment of advanced-stage melanoma harboring BRAF mutations. However, drug resistance, through mechanisms not well elucidated, often occurs. A better understanding of how melanoma-derived immunologically active molecules change in response to MAPK inhibition of BRAF mutated (BRAF) and BRAF wild type (BRAF) melanomas could help identify promising treatment combinations of small molecule inhibitors and immunotherapy. To this aim, we treated 13 BRAF and 13 BRAF mutated human melanoma cell lines with either a specific BRAF inhibitor or an MEK1/2 inhibitor and analyzed changes in the secretion of 42 selected cytokines, chemokines, and growth factors. We also measured changes in the expression levels of immunologically relevant melanoma cell surface markers. The BRAF melanomas showed minimal changes in response to the inhibitors, whereas the BRAF cell lines showed, on average, a significant decrease in IFNα2, interleukin-7, Fractalkine, GCSF, GRO, TGFα2, interleukin-8, and VEGF, as well as a reduction in pERK and pMEK protein levels, upon MAPK pathway blockade. BRAF inhibition in BRAF cell lines also resulted in significant changes in the expression of several surface markers including upregulation of ß2-microglobulin as well as a decrease in MIC A/B and TRAIL-R2. These results indicate that MAPK pathway inhibition leads to changes in the immunological properties of mutant BRAF melanoma cells and lends support for future studies aimed at designing effective treatment strategies that combine BRAF and MEK inhibition with immunotherapy.

Clinical and immunologic effects of intranodal autologous tumor lysate-dendritic cell vaccine with Aldesleukin (Interleukin 2) and IFN-{alpha}2a therapy in metastatic renal cell carcinoma patients.

PURPOSE: To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-alpha 2a in metastatic renal cell carcinoma patients. EXPERIMENTAL DESIGN: Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 x 10(7) cells/1 mL Lactated Ringer's solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-alpha 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti-renal cell carcinoma antibody and cytokine levels. RESULTS: All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4(+) T regulatory cells had a strong association with outcome. Pre-IP-10 serum levels approached significance for predicting outcome. CONCLUSIONS: The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.

Ex vivo expansion of tumor specific lymphocytes with IL-15 and IL-21 for adoptive immunotherapy in melanoma.

Although T central memory cells have been described as the most effective T-cell subtype against tumor growth, little is known about the requirements needed for their optimal ex vivo generation. Hence, our goal is to establish a protocol that will lead to consistent ex vivo generation of lymphocytes skewed toward a central memory phenotype. Antigen-specific T-cell lines were generated by ex vivo stimulation with Class-I and Class-II melanoma peptide pulsed dendritic cells in the presence of either IL-2 or IL-15 plus IL-21. Tumor specific lymphocytes of both central memory and effector characteristics were consistently generated from healthy donors and melanoma patients. IL15/IL21 cultures result in a cell population with a lower proportion of CD4(+)CD25(high)FoxP3(+) regulatory cells and higher number of CD8(+) and CD56(+) cells, and consequently render a higher yield of cells with a greater cytolytic activity and IFN-gamma production against melanoma cell lines.

Developing a rational tumor vaccine therapy for renal cell carcinoma: immune yin and yang.

In patients with progressive malignancy, the natural balance between proinflammatory (Yang) and inhibitory (regulatory or Yin) immune pathways is disrupted and favors cancer-specific immune suppression. Therapy with interleukin 2 (IL-2) can mobilize immune effector cells that recognize and destroy cancer. High-dose IL-2 is the only therapy that has consistently induced complete durable remissions in patients with metastatic renal cell carcinoma (RCC) but only in a few of them. The lack of benefit in most metastatic RCC patients is likely due to the ineffective manipulation of other immune circuits critical in regulating tumor cytotoxic pathways. The limited clinical activity of IL-2, RCC vaccines, and other immune therapies to date leads us to postulate that effective clinical treatment strategies will need to simultaneously enhance proinflammatory pathways and disrupt regulatory pathways. We present preliminary studies in RCC patients to highlight the complexity of the regulatory pathways and our approach to shifting the balance of proinflammatory and regulatory immune pathways using dendritic cell-tumor lysate vaccine followed by cytokine therapy.