Limper, Andrew H.

Abstract Pneumocystis species interaction with myeloid cells is well known, especially in macrophages; however, how the organism binds to lung epithelial cells is incompletely understood. Ephrin type-A receptor 2 (EphA2) has been previously identified as a lung epithelial pattern recognition receptor that binds to fungal β-glucans. Herein, we also report that EphA2 can also bind Pneumocystis β-glucans, both in isolated forms and also on exposed surfaces of the organism. Furthermore, binding of Pneumocystis β-glucans resulted in phosphorylation of the EphA2 receptor, which has been shown to be important for downstream proinflammatory response. Indeed, we also show that interleukin 6 cytokine is significantly increased when lung epithelial cells are exposed to Pneumocystis β-glucans, and that this response could be blocked by preincubation with a specific antibody to EphA2. Our study presents another Pneumocystis lung epithelial cell receptor with implications for initial colonization and possible therapeutic intervention.

AbstractBackgroundPneumocystis jirovecii pneumonia (PJP) is a life‐threatening infection occurring in patients receiving bendamustine. The poorly defined incidence, particularly when utilizing polymerase chain reaction (PCR)‐based diagnostic techniques, precipitates unclear prophylaxis recommendations. Our objective was to determine the cumulative incidence of PJP diagnosed by single copy target, non‐nested PCR in patients receiving bendamustine.MethodsPatients were evaluated for PJP from initiation of bendamustine through 9 months after the last administration. The cumulative incidence of PJP was estimated using the Aalen–Johansen method. Cox proportional hazard models were used to demonstrate the strength of association between the independent variables and PJP risk.ResultsThis single‐center, retrospective cohort included 486 adult patients receiving bendamustine from 1 January 2006 through 1 August 2019. Most patients received bendamustine‐based combination therapy (n = 461, 94.9%), and 225 (46.3%) patients completed six cycles. Rituximab was the most common concurrent agent (n = 431, 88.7%). The cumulative incidence of PJP was 1.7% (95% CI 0.8%–3.3%, at maximum follow‐up of 2.5 years), after the start of bendamustine (n = 8 PJP events overall). Prior stem cell transplant, prior chemotherapy within 1 year of bendamustine, and lack of concurrent chemotherapy were associated with the development of PJP in univariate analyses. Anti‐Pneumocystis prophylaxis was not significantly associated with a reduction in PJP compared to no prophylaxis (HR 0.37, 95% CI (0.05, 3.04), p = 0.36).ConclusionsOur incidence of PJP below 3.5%, the conventional threshold for prophylaxis implementation, indicates routine anti‐Pneumocystis prophylaxis may not be necessary in this population. Factors indicating a high‐risk population for targeted prophylaxis require further investigation.

Pneumocystis, an opportunistic fungus, causes pneumonia in immunocompromized hosts. Inflammation is induced, at least partly, by β‐eta‐glucan, the major component of Pneumocystis cell wall. Airspace‐lining epithelial cells recognize glucan by membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer). We treated Pneumocystis‐infected mice with two GSL inhibitors: PDMP and NB‐DNJ (Miglustat‐Actelion Pharmaceuticals). Starting at 8 weeks post infection, animals received either 150 mg PDMP or 1200 mg Miglustat/kg of body weight ‐ dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). Results show that PDMP‐treated mice had fewer leukocytes in bronchoalveolar lavage fluid (BALF) than saline‐treated controls, with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP‐2 and TNF‐alpha (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q‐PCR, showed decrease in PDMP‐treated animals. The anti‐inflammatory effects of Miglustat were less pronounced than PDMP effects. Neither inhibitor induced lung injury as measured by albumin leak into airspaces. Results suggest that GSL inhibitors might have an impact on inflammatory response to Pneumocystis, leading to new options in treatment of Pneumocystis pneumonia. Supported by NIH‐RO1‐HL62150 and Mayo Clinic

Abstract BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease characterized by aberrant tissue remodeling, formation of scar tissue within the lungs and continuous loss of lung function. The areas of fibrosis seen in lungs of IPF patients share many features with normal aging lung with cellular senescence being one. The contribution of the immune system to the etiology of IPF remains poorly understood. Evidence obtained from animal models and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. Currently, there is only modestly effective pharmacotherapy for IPF. Mesenchymal stem cells (MSCs)-based therapies have emerged as a potential option treatment of IPF. This study explores the possibility of using autologous MSCs as an IPF therapy and present an attempt to determine if the disease occurring in the lungs can be reflected on peripheral blood immune status. MethodsComprehensive characterization of autologous adipose derived MSCs (aMSCs) from five IPF patients and five age and gender matched Healthy Controls (HC) was done using flow cytometry, Droplet Digital PCR (ddPCR), Multiplex Luminex xMAP technology and confocal microscopy. For assessing the self renewal capacity and osteogenic differentiation IncuCyte Live Cell Imaging technology was used. Multi-parameter quantitative flow cytometry of un-manipulated whole blood of another group of 15 IPF patients and 87 (30 age and gender matched) HC was used to analyze 110 peripheral phenotypes.ResultsThere are no differences between autologous aMSCs from IPF patients and HC in their stem cell properties, self renewal capacity, plasticity for osteogenic differentiation, secretome content, cell cycle inhibitor marker levels and mitochondrial health. IPF patients had altered peripheral blood immunophenotype including reduced B cells subsets, increased T cell subsets, and increased granulocytes among others demonstrating clear and significant differences.ConclusionsOur results indicate that there is no difference in aMSCs properties from IPF patients and HC, suggesting that autologous aMSCs may be an acceptable option for IPF therapy.Characterization of peripheral immune phenotype may be a valuable indicator for successful therapy, and for potentially staging the disease.

Pneumocystis jirovecii is a fungal pathogen that can cause life-threatening infections in individuals who are immunocompromised. Acquired via inhalation, upon entering the respiratory tract, the fungi first encounter innate immune cells such as alveolar macrophages (AMs). Relatively little is known about the AM cellular responses to the organism, and particularly transcription factor (TF) profiles leading to early host responses during infection. Utilizing the Mouse Transcription Factors RT2 Profiler™ PCR Array, we report an initial TF survey of these macrophage and Pneumocystis interactions. Expression levels of a panel of mouse TFs were compared between unstimulated and Pneumocystis murina-stimulated AMs. Interestingly, a number of TFs previously implicated in pathogen–host cell interactions were highly up- or downregulated, including hif1a and Pparg. qPCR experiments were further conducted to verify the results of these surveyed transcripts. Furthermore, with immunoblotting, we show that HIF-1A and PPAR-γ are indeed significantly upregulated and downregulated, respectively. Lastly, and importantly, we report that in the mouse model of Pneumocystis pneumonia (PCP), which mimics human Pneumocystis jirovecii pneumonia (PJP), qPCR analysis of Pneumocystis murina lungs also mimic the initial TF profile analysis, suggesting an importance for these TFs in immunocompromised hosts with Pneumocystis pneumonia. These data demonstrate the use of TF profiling in host AMs and Pneumocystis organism interactions that may lead to a better understanding of the specific inflammatory responses of the host to Pneumocystis pneumonia and may inform novel strategies for potential therapeutics.

Objectives Evaluate associations between ACE inhibitors (ACEis) and angiotensin receptor blockers (ARBs) and clinical outcomes in acute viral respiratory illness (AVRI). Design Retrospective cohort analysis of claims data. Setting The USA; 2018–2019 influenza season. Participants Main cohort: people with hypertension (HTN) taking an ACEi, ARB or other HTN medications, and experiencing AVRI. Falsification cohort: parallel cohort receiving elective knee or hip replacement. Main outcome measures Main cohort: hospital admission, intensive care unit, acute respiratory distress (ARD), ARD syndrome and all-cause mortality. Falsification cohort: complications after surgery and all-cause mortality. Results The main cohort included 236 843 episodes of AVRI contributed by 202 629 unique individuals. Most episodes were in women (58.9%), 81.4% in people with Medicare Advantage and 40.3% in people aged 75+ years. Odds of mortality were lower in the ACEi (0.78 (0.74 to 0.83)) and ARB (0.64 (0.61 to 0.68)) cohorts compared with other HTN medications. On all other outcomes, people taking ARBs (but not ACEis) had a >10% reduction in odds of inpatient stays compared with other HTN medications. In the falsification analysis (N=103 353), both ACEis (0.89 (0.80 to 0.98)) and ARBs (0.82 (0.74 to 0.91)) were associated with decreased odds of complications compared with other HTN medications; ARBs (0.64 (0.47 to 0.87)) but not ACEis (0.79 (0.60 to 1.05)) were associated with lower odds of death compared with other HTN medications. Conclusions Outpatient use of ARBs was associated with better outcomes with AVRI compared with other medications for HTN. ACEis were associated with reduced risk of death, but with minimal or no reduction in risk of other complications. A falsification analysis conducted to provide context on the possible causal implications of these findings did not provide a clear answer. Further analysis using observational data will benefit from additional approaches to assess causal relationships between these drugs and outcomes in AVRI.

Abstract B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and β-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell–secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell–mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

Abstract Rationale In October 2014, the antifibrotic medications pirfenidone and nintedanib became the first medications approved by the U.S. Food and Drug Administration for use in patients with idiopathic pulmonary fibrosis (IPF). Since approval, there has been no nonregistry analysis of the real-world adoption of these medications in everyday clinical practice. Objectives To evaluate the adoption, persistence, and out-of-pocket (OOP) costs of pirfenidone and nintedanib since their approval in the United States in 2014. Methods A retrospective cohort analysis was performed by identifying privately insured and Medicare Advantage beneficiaries with IPF. We then split the patients into three cohorts: those who were untreated and those who filled a prescription for either pirfenidone or nintedanib between October 1, 2014, and July 31, 2019. The primary outcome was adoption of the medications. Secondary outcomes included medication persistence and prescription drug costs. Results A total of 10,996 patients with IPF were identified in the data set. A minority of patients (26.4%) with IPF identified in the cohort had started either medication since approval in 2014, with the adoption of both medications being comparable at around 13.2%. Those receiving the medications were younger (72 vs. 73.9 yr; P < 0.0001) and healthier (3.9 vs. 4.9 comorbidities; P < 0.0001) than those not receiving treatment. Men were significantly more likely to receive treatment than woman (30.0% vs. 21.9%; P < 0.0001). Among treated patients, 42.8% discontinued the medications during the study period. Patients’ OOP expenses per month were high for both drugs (mean, $397.51 for nintedanib; mean, $394.49 for pirfenidone). Conclusions The adoption of both the antifibrotic medications in the United States in everyday practice has been low since approval and may be associated with the high OOP cost.