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BACKGROUND: In children, the acute pyelonephritis that can result from urinary tract infections (UTIs), which commonly ascend from the bladder to the kidney, is a growing concern because it poses a risk of renal scarring and irreversible loss of kidney function. To date, the cellular mechanisms underlying acute pyelonephritis–driven renal scarring remain unknown. METHODS: We used a preclinical model of uropathogenic Escherichia coli–induced acute pyelonephritis to determine the contribution of neutrophils and monocytes to resolution of the condition and the subsequent development of kidney fibrosis. We used cell-specific monoclonal antibodies to eliminate neutrophils, monocytes, or both. Bacterial ascent and the cell dynamics of phagocytic cells were assessed by biophotonic imaging and flow cytometry, respectively. We used quantitative RT-PCR and histopathologic analyses to evaluate inflammation and renal scarring. RESULTS: We found that neutrophils are critical to control bacterial ascent, which is in line with previous studies suggesting a protective role for neutrophils during a UTI, whereas monocyte-derived macrophages orchestrate a strong, but ineffective, inflammatory response against uropathogenic, E. coli–induced, acute pyelonephritis. Experimental neutropenia during acute pyelonephritis resulted in a compensatory increase in the number of monocytes and heightened macrophage-dependent inflammation in the kidney. Exacerbated macrophage-mediated inflammatory responses promoted renal scarring and compromised renal function, as indicated by elevated serum creatinine, BUN, and potassium. CONCLUSIONS: These findings reveal a previously unappreciated outcome for neutrophil-macrophage imbalance in promoting host susceptibility to acute pyelonephritis and the development of permanent renal damage. This suggests targeting dysregulated macrophage responses might be a therapeutic tool to prevent renal scarring during acute pyelonephritis.

Background Cystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response. Methods Whole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses. Results We identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug. Conclusions Transcriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.

Background There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes. Methods Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds. Results AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells. Conclusions The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.

During infection, phagocytic cells pursue homeostasis in the host via multiple mechanisms that control microbial invasion. Neutrophils respond to infection by exerting a variety of cellular processes, including chemotaxis, activation, phagocytosis, degranulation and the generation of reactive oxygen species (ROS). Calcium (Ca2+) signaling and the activation of specific Ca2+ channels are required for most antimicrobial effector functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating Ca2+ mobilization and oxidative stress in neutrophils. In the present study, we use a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils' functions during infection. We show that the susceptibility of Trpm2−/− mice to L. monocytogenes infection is characterized by increased migration rates of neutrophils and monocytes to the liver and spleen in the first 24 h. During the acute phase of L. monocytogenes infection, Trpm2−/− mice developed septic shock, characterized by increased serum levels of TNF-α, IL-6, and IL-10. Furthermore, in vivo depletion of neutrophils demonstrated a critical role of these immune cells in regulating acute inflammation in Trpm2−/− infected mice. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2−/− neutrophils. Finally, the increased inflammatory properties of Trpm2−/− neutrophils correlated with the dysregulated cytoplasmic concentration of Ca2+ and potentiated membrane depolarization, in response to L. monocytogenes. In conclusion, our findings suggest that the TRPM2 channel plays critical functional roles in regulating the inflammatory properties of neutrophils and preventing tissue damage during Listeria infection.

Nontypeable Haemophilus influenzae (NTHI) is the causative agent of multiple respiratory tract infections. Several human pathogens, including NTHI, possess a novel genetic system, termed the phasevarion, which mediates a rapid and reversible change in the expression of many genes throughout the chromosome. This occurs by phase variation of a single gene (modA) that encodes a DNA methyltransferase and results in two phenotypically distinct subpopulations, ON and OFF. NTHI encounters many pressures within the various microenvironments of its human host as the disease course evolves from one of asymptomatic nasopharyngeal carriage to overt disease. These include oxidative stresses, which are present throughout the respiratory tract. To persist in the human nasopharynx and as a pathogen throughout the airways, NTHI must be able to mitigate toxic levels of oxidative stress. Here we show that expression of ModA2, modA2 ON status, resulted in increased sensitivity to oxidative stress. Furthermore, the modA2 ON status resulted in decreased resistance to neutrophil-mediated killing, which resulted in selection for the modA2 OFF subpopulation in an ex vivo survival assay. These findings highlight the importance of the ModA2 phasevarion in adaptation to innate host defences and reveal an additional microenvironmental pressure that selected for a specific ModA2 subpopulation.

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/β leading to reduced phosphorylation of NADPH oxidase components p47phox and p40phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia. These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1β and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11 -/- mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1β, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1β induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11 -/- neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11 -/- neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

The staphylococcal α-hemolysin is critical for the pathogenesis of Staphylococcus aureus skin and soft tissue infection. Vaccine and infection-elicited α-hemolysin–specific antibodies protect against S. aureus–induced dermonecrosis, a key feature of skin and soft tissue infection. Many interactions between α-hemolysin and host cells have been identified that promote tissue damage and modulate immune responses, but the mechanisms by which protective adaptive responses cross talk with innate responses at the tissue level are not clear. Using an established mouse model of skin and soft tissue infection and a newly developed histopathologic scoring system, we observed pathologic correlates early after infection, predicting protection against dermonecrosis by anti–α-hemolysin antibody. Protection was characterized by robust neutrophilic inflammation and compartmentalization of bacteria into discrete abscesses, which led to the attenuation of dermonecrosis and enhancement of bacterial clearance later in the infection. The ultimate outcome of infection was driven by the recruitment of neutrophils within the first day after infection but not later. Antibody-mediated protection was dependent on toxin neutralization rather than on enhanced opsonophagocytic killing by neutrophils or protection against toxin-mediated neutrophil lysis. Together, these findings advance our understanding of the mechanisms by which the early synergism between antibody-mediated toxin neutralization and tissue-specific neutrophilic inflammation preserve tissue integrity during infection.

We previously developed a tissue-engineered vascular graft (TEVG) made by seeding autologous cells onto a biodegradable tubular scaffold, in an attempt to create a living vascular graft with growth potential for use in children undergoing congenital heart surgery. Results of our clinical trial showed that the TEVG possesses growth capacity but that its widespread clinical use is not yet advisable due to the high incidence of TEVG stenosis. In animal models, TEVG stenosis is caused by increased monocytic cell recruitment and its classic (“M1”) activation. Here, we report on the source and regulation of these monocytes. TEVGs were implanted in wild-type, CCR2 knockout (Ccr2(−/−)), splenectomized, and spleen graft recipient mice. We found that bone marrow–derived Ly6C(+hi) monocytes released from sequestration by the spleen are the source of mononuclear cells infiltrating the TEVG during the acute phase of neovessel formation. Furthermore, short-term administration of losartan (0.6 g/L, 2 wk), an angiotensin II type 1 receptor antagonist, significantly reduced the macrophage populations (Ly6C(+/−)/F480(+)) in the scaffolds and improved long-term patency in TEVGs. Notably, the combined effect of bone marrow–derived mononuclear cell seeding with short-term losartan treatment completely prevented the development of TEVG stenosis. Our results provide support for pharmacologic treatment with losartan as a strategy to modulate monocyte infiltration into the grafts and thus prevent TEVG stenosis.—Ruiz-Rosado, J. D. D., Lee, Y.-U., Mahler, N., Yi, T., Robledo-Avila, F., Martinez-Saucedo, D., Lee, A. Y., Shoji, T., Heuer, E., Yates, A. R., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr−/−, human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia. Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

INTRODUCTION: Cystic fibrosis (CF) is a multi-organ disorder characterized by chronic sino-pulmonary infections and inflammation. Many patients with CF suffer from repeated pulmonary exacerbations that are predictors of worsened long-term morbidity and mortality. There are no reliable markers that associate with the onset or progression of an exacerbation or pulmonary deterioration. Previously, we found that the Mirc1/Mir17–92a cluster which is comprised of 6 microRNAs (Mirs) is highly expressed in CF mice and negatively regulates autophagy which in turn improves CF transmembrane conductance regulator (CFTR) function. Therefore, here we sought to examine the expression of individual Mirs within the Mirc1/Mir17–92 cluster in human cells and biological fluids and determine their role as biomarkers of pulmonary exacerbations and response to treatment. METHODS: Mirc1/Mir17–92 cluster expression was measured in human CF and non-CF plasma, blood-derived neutrophils, and sputum samples. Values were correlated with pulmonary function, exacerbations and use of CFTR modulators. RESULTS: Mirc1/Mir17–92 cluster expression was not significantly elevated in CF neutrophils nor plasma when compared to the non-CF cohort. Cluster expression in CF sputum was significantly higher than its expression in plasma. Elevated CF sputum Mirc1/Mir17–92 cluster expression positively correlated with pulmonary exacerbations and negatively correlated with lung function. Patients with CF undergoing treatment with the CFTR modulator Ivacaftor/Lumacaftor did not demonstrate significant change in the expression Mirc1/Mir17–92 cluster after six months of treatment. CONCLUSIONS: Mirc1/Mir17–92 cluster expression is a promising biomarker of respiratory status in patients with CF including pulmonary exacerbation. Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society.

Helminths cause chronic infections and affect the immune response to unrelated inflammatory diseases. Although helminths have been used therapeutically to ameliorate inflammatory conditions, their anti-inflammatory properties are poorly understood. Alternatively activated macrophages (AAMϕs) have been suggested as the anti-inflammatory effector cells during helminth infections. Here, we define the origin of AAMϕs during infection with Taenia crassiceps, and their disease-modulating activity on the Experimental Autoimmune Encephalomyelitis (EAE). Our data show two distinct populations of AAMϕs, based on the expression of PD-L1 and PD-L2 molecules, resulting upon T. crassiceps infection. Adoptive transfer of Ly6C+ monocytes gave rise to PD-L1+/PD-L2+, but not PD-L1+/PD-L2− cells in T. crassiceps-infected mice, demonstrating that the PD-L1+/PD-L2+ subpopulation of AAMϕs originates from blood monocytes. Furthermore, adoptive transfer of PD-L1+/PD-L2+ AAMϕs into EAE induced mice reduced disease incidence, delayed disease onset, and diminished the clinical disability, indicating the critical role of these cells in the regulation of autoimmune disorders.

Macrophage migration inhibitory factor (MIF) mediates immunity againstToxoplasma gondiiinfection by inducing inflammatory cytokines required to control the parasite replication. However, the role of this inflammatory mediator in the cell-mediated immune response against this infection is still poorly understood. Here, we usedT. gondii-infected WT andMif−/−mice to analyze the role of MIF in the maturation of CD11b+and CD8α+dendritic cells (DCs). We found that MIF promotes maturation of CD11b+but not CD8α+DCs, by inducing IL-12p70 production and CD86 expression. InfectedMif−/−mice showed significantly lower numbers of TNF and inducible nitric oxide synthase- (iNOS-) producing DCs (TipDCs) compared to infected WT mice. The adoptive transfer ofLy6Chighmonocytes into infected WT orMif−/−mice demonstrated that MIF participates in the differentiation ofLy6Chighmonocytes into TipDCs. In addition, infectedMif−/−mice display a lower percentage of IFN-γ-producing natural killer (NK) cells compared to WT mice, which is associated with reducing numbers of TipDCs inMif−/−mice. Furthermore, administration of recombinant MIF (rMIF) intoT. gondii-infectedMif−/−mice restored the numbers of TipDCs and reversed the susceptible phenotype ofMif−/−mice. Collectively, these results demonstrate an important role for MIF inducing cell-mediated immunity toT. gondiiinfection.

Stenosis is a critical problem in the long-term efficacy of tissue-engineered vascular grafts (TEVGs). We previously showed that host monocyte infiltration and activation within the graft drives stenosis and that TGF-β receptor 1 (TGF-βR1) inhibition can prevent it, but the latter effect was attributed primarily to inhibition of mesenchymal cell expansion. In this study, we assessed the effects of TGF-βR1 inhibition on the host monocytes. Biodegradable TEVGs were implanted as inferior vena cava interposition conduits in 2 groups of C57BL/6 mice (n = 25/group): unseeded grafts and unseeded grafts with TGF-βR1 inhibitor systemic treatment for the first 2 wk. The TGF-βR1 inhibitor treatment effectively improved TEVG patency at 6 mo compared to the untreated control group (91.7 vs. 48%, P < 0.001), which is associated with a reduction in classic activation of mononuclear phagocytes. Consistent with these findings, the addition of rTGF-β to LPS/IFN-γ-stimulated monocytes enhanced secretion of inflammatory cytokines TNF-α, IL-12, and IL-6; this effect was blocked by TGF-βR1 inhibition (P < 0.0001). These findings suggest that the TGF-β signaling pathway contributes to TEVG stenosis by inducing classic activation of host monocytes. Furthermore, blocking monocyte activation by TGF-βR1 inhibition provides a viable strategy for preventing TEVG stenosis while maintaining neotissue formation.-Lee, Y.-U., de Dios Ruiz-Rosado, J., Mahler, N., Best, C. A., Tara, S., Yi, T., Shoji, T., Sugiura, T., Lee, A. Y., Robledo-Avila, F., Hibino, N., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. TGF-β receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation.