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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.

Patients with Cystic Fibrosis (CF) are highly susceptible to infections caused by opportunistic pathogens including Burkholderia cenocepacia. In response to infection, CF neutrophils are deficient in clearing bacteria and release inflammatory molecules that contributes to aggravated lung-tissue damage in people with CF (PWCF). Although CF has no cure, novel therapeutic drugs have emerged targeting defective CFTR channel (CFTR modulators). However, it is unknown whether CFTR modulators may restore the basic antimicrobial functions of phagocytes in PWCF. Here, we first evaluated the expression and subcellular localization of CFTR. Neutrophils from PWCF and non-CF donors were treated in vitro with CF modulators, before infection with B. cenocepacia, and then, CFTR cellular localization was evaluated by confocal microscopy followed by the analysis of antimicrobial killing, and the production of Neutrophil Extracellular Traps (NETs). Furthermore, CFTR-mediated currents were measured by patch-clamp in non-CF and CF neutrophils after treatment with the CFTR modulators. We found that CFTR modulators increased the expression of functional CFTR channel at the plasma membrane, and also potentiated CFTR-mediated channel activity in CF and non-CF neutrophils, as recorded by whole-cell patch clamp. The production of NETs was also increased in both CF and non-CF neutrophils treated with CFTR modulators, those changes were comparable to cells stimulated with B. cenocepacia or PMA. Finally, CF neutrophils treated with the CFTR modulators restored their antimicrobial killing against B. cenocepacia. Together, these data indicate that functional CFTR channel is essential for regulating the antimicrobial mechanisms of neutrophils.

Every year, pediatric urinary tract infections (UTI) account for 1.5 million clinician visits in the US alone. Up to 50% of infants with UTI develop a kidney infection (acute pyelonephritis, APN). Despite antibiotic therapy, 15% of children with APN develop renal scarring, which is associated with deterioration in kidney function. Here, we determined the contribution of macrophages and neutrophils to bacterial clearance and the development of kidney fibrosis during experimental APN. Female C3H/HeOuJ mice were treated, via retro-orbital, with one of the following antibodies: isotype IgG (control), anti-Ly6G (depletes neutrophils), anti-CD115 (depletes monocytes), and anti-GR1 (depletes monocytes and neutrophils). After cell depletion, the animals were transurethrally infected with UPEC. Ascending UTI was evaluated via biophotonic imaging. The dynamics of monocytes and neutrophils were determined by flow cytometry. Histopathological scores were performed by H&E and Sirius-Red stainings. Transcriptomic analyses of kidneys were performed using TaqMan Arrays. Our data indicate that neutrophils are required for bacterial clearance, while macrophages promote inflammation in the infected urinary tract. However, the absence of neutrophils results in increased macrophage infiltration and exaggerated macrophage-dependent inflammatory responses in the bladder and kidney. Also, our results demonstrate that macrophage-dependent inflammation during APN, leads to renal scarring and renal functional impairment. These findings uncover a role for the neutrophil-macrophage imbalance in promoting kidney fibrosis during APN. This knowledge will be helpful to develop novel therapies to prevent post-APN scarring in children.

Calcium signaling is required for most antimicrobial functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating calcium mobilization, cell migration and oxidative stress in neutrophils. However, the precise mechanism by which TRPM2 activation may exert a pro- or anti-inflammatory function remains unclear. In this study, we used a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils’ function. We found that infected Trpm2−/− mice were associated with high bacterial burden in spleen and liver indicating increased susceptibility to Listeria infection. Pronounced migration rates of neutrophils and monocytes to the liver and spleen characterized the inflammatory response to Listeria during the first 18h. In the Trpm2−/− mice, the acute phase infection resulted in septic shock, defined by increased serum levels of TNF-α, IL-6, and IL-10, but no decreased levels of IFN-γ or IL-12. Furthermore, depletion of neutrophils demonstrated the critical role of these cells in regulating acute inflammation by conferring resistance of Trpm2−/− mice to Listeria infection. 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 cellular calcium overloading and potentiated membrane depolarization, in response to Listeria infection. In conclusion, our findings suggest that TRPM2 channel plays critical roles in the regulation of the inflammatory properties of neutrophils during Listeria monocytogenes infection.

Monocytes and macrophages express Fc-gamma Receptors (FcγR) that engage IgG immune complexes such as antibody-opsonized pathogens or cancer cells to destroy them by various mechanisms including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors such as FcγRIIb and SIRPα. Here, we report that another ITIM-containing receptor, Platelet Endothelial Adhesion Molecule-1 (PECAM1/CD31), regulates FcγR function, and is itself regulated by FcγR activation. First, qRT-PCR and flow cytometry analyses revealed that monocyte FcγR activation leads to significant downregulation of CD31 expression, both at the message level and at surface expression. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that loss of surface expression was due to internalization, which depended predominantly on the PI3 Kinase pathway. Finally, functional analyses showed that downregulation of CD31 expression in monocytes by siRNA enhanced FcγR-mediated phagocytic ability. Together, these results suggest that CD31 functions as a checkpoint receptor that could be targeted to enhance FcγR functions in antibody-mediated therapies.

Cyclic ADP ribose (cADPR) is a calcium-mobilizing metabolite that regulates intracellular calcium release and extracellular calcium influx. Although the role of cADPR in modulating calcium mobilization has been extensively examined, its potential role in regulating immunologic responses is less well understood. We previously reported that cADPR, produced by the ADP-ribosyl cyclase, CD38, controls calcium influx and chemotaxis of murine neutrophils responding to fMLF, a peptide agonist for two chemoattractant receptor subtypes, formyl peptide receptor and formyl peptide receptor-like 1. In this study, we examine whether cADPR is required for chemotaxis of human monocytes and neutrophils to a diverse array of chemoattractants. We found that a cADPR antagonist and a CD38 substrate analogue inhibited the chemotaxis of human phagocytic cells to a number of formyl peptide receptor-like 1-specific ligands but had no effect on the chemotactic response of these cells to ligands selective for formyl peptide receptor. In addition, we show that the cADPR antagonist blocks the chemotaxis of human monocytes to CXCR4, CCR1, and CCR5 ligands. In all cases, we found that cADPR modulates intracellular free calcium levels in cells activated by chemokines that induce extracellular calcium influx in the apparent absence of significant intracellular calcium release. Thus, cADPR regulates calcium signaling of a discrete subset of chemoattractant receptors expressed by human leukocytes. Since many of the chemoattractant receptors regulated by cADPR bind to ligands that are associated with clinical pathology, cADPR and CD38 represent novel drug targets with potential application in chronic inflammatory and neurodegenerative disease.

Helicobacter pylori (Hp) is a dominant member of the gastric microbiota in a majority of the world’s population. Hp colonization has been attributed to detrimental outcomes including gastritis and gastric cancer. The immune response to Hp leads to elevated levels of reactive oxygen species and reactive nitrogen species within the gastric mucosa. The redox-sensitive cation channel TRPM2 is activated by direct binding of ADP ribose and under conditions of oxidative and metabolic stress by H2O2 and H+. TRPM2 is strongly expressed in immune cells of the phagocytic lineage, themselves professional oxidants-generators. Here we report that Hp infection of TRPM2-deficient macrophages resulted in higher rates of bacterial killing and increased levels of proinflammatory cytokines and NOX2 compared to wild-type macrophages. Consequently, TRPM2-deficient macrophages displayed significantly higher ROS production during Hp infection, suggesting an increased NADPH oxidase activity. In vivo, when TRPM2-deficient mice were infected orogastrically with Hp their gastric tissues exhibited an increased NOX2 expression and myeloperoxidase activity compared to wild-type mice. Furthermore, enhancement of NADPH oxidase activity in TRPM2-deficient animals increased inflammation and reduced bacterial burden when compared with wild-type mice. Taken together, these data indicate that TRPM2 establishes a negative feedback of ROS production through phagosomal NADPH oxidase during Hp infection.

Phagocytes protect the urinary tract from bacterial infections (UTI). Neutrophils are required early upon infection for clearing off bacteria, however much less is known regarding the function of monocyte-derived macrophages during UTI. Two main subsets of blood monocytes can be distinguished by differential expression of the chemokine receptors CCR2 and CX3CR1. To determine the functional role of monocyte-derived phagocytes, we used CX3CR1 and CCR2 deficient mice and monitored the trafficking of monocyte-derived macrophages during the course of uropathogenic Escherichia coli (UPEC) induced UTI. We found that Ly6+hi/CCR2+hi/CX3CR1+low, classical inflammatory monocytes were recruited from the blood to the bladder and kidney at 8-24h after infection. A subset of Ly6+low/CCR2+low/CX3CR1+hi nonclassical blood monocytes was recruited to urinary organs early (4h) after infection. UPEC induced bacterial burden was significantly elevated in the bladder of CCR2 KO but not CX3CR1 KO mice at 16h post-infection, notoriously, CX3CR1 KO mice were unable to clear UPEC at 48h post-infection; instead, bacteria spread from the bladder to the kidney causing pyelonephritis in these mice but not in WT controls. Our studies indicate that classical monocyte derived macrophages are required to clear bacteria from the bladder during early UTI infection, in contrast, nonclassical macrophages are required for preventing later bacterial spreading, and likely the clearance of bacteria in the kidney.

Blood monocyte-derived macrophages (MØ) and dendritic cells (DCs) are essential for effective control of infections. Exacerbated recruitment of these cells, in the other side, contributes to the pathogenesis of chronic inflammatory diseases. The main blood monocyte subsets differ in the expression of the differentiation marker Ly6C and the chemokine receptors CCR2 and CX3CR1. We recently found a subset of Ly6Clow/CCR2low/CX3CR1hi associated to inflammatory responses induced by urinary tract infection in the mouse. Therefore, here we analyzed monocyte-derived inflammatory cell subsets in a model of non-bacterial peritonitis using CCR2 and CX3CR1 KO mice. We found that CCR2 is not required for tissue recruitment of monocyte-derived MØ under homeostatic conditions. In contrast, CCR2 expression is required for the recruitment of two Ly6Chi/CX3CR1low and Ly6Clow/CX3CR1hi subpopulations of inflammatory cells. Furthermore, sorted Ly6Clow/CX3CR1hi cells produced high levels of IL-6, MIP-1 and TNF-α cytokines. These cells showed dendritic cell morphology, low levels of F4/80 but CD11c expression, as well as Class-II, CD80, and CD86 DC markers. In addition, Ly6Clow/CX3CR1hi cells were capable of picking-up antigen and trafficked to lymph nodes demonstrating specific DC functions. In summary, our studies indicate that blood monocytes expressing Ly6Clow/CX3CR1hi give rise to a novel inflammatory DC subset recruited to tissue during infectious or non-infectious inflammatory conditions.

Phagocytes are essential for protecting the urinary tract from bacterial infections. Although neutrophils are required for clearing of bacteria during urinary tract infection (UTI), the function of monocyte-derived macrophages and dendritic cells (DCs) is less clear. To determine the role of monocyte-derived phagocytes, we used CD11b-DTRTg mice and selectively depleted CD11b+ blood monocytes prior to inoculation of uropathogenic Escherichia coli (UPEC) into the bladder. We then monitored monocyte-derived macrophages and DCs during the course of UTI. We found that conditional CD11b ablation effectively depleted a population of CXC3CR1+high, but not CX3CR1+low cells in the bladder and kidney. Interestingly, CX3CR1+high cells expressed low levels of F4/80 but expressed CD11c, and other DC markers, suggesting they were monocyte-derived DCs. UPEC induced bacterial burden was significantly elevated in the kidneys of CD11b-depleted mice at 48 hrs post-infection, when compared to controls. Furthermore, CX3CR1 KO mice were unable to clear UPEC 48 hrs post-infection; instead, bacteria spread from the bladder to the kidney, similarly to CD11b monocyte depleted mice. CX3CR1+high DCs, but not CX3CR1+low macrophages isolated from infected bladders produced abundant amounts of IL-6 and TNF-α pro-inflammatory cytokines. Our studies indicate that a monocyte-derived novel DC subset expressing CX3CR1 is required for preventing bacterial spreading, and the clearance of bacteria in the kidney.

Our current understanding of how T lymphocytes enter the airways in response to respiratory tract infections is limited. Here we show a role for type I interferon (IFN) signaling in regulating T lymphocyte transmigration into the airway mucosa. IFNα receptor (IFNAR) deficiency led to a reduction in the number of T lymphocytes that entered the airways but not the lung parenchyma. This effect was mediated both at the T lymphocyte and epithelial cell level. IFNAR-deficient airway epithelial cells failed to express the chemoattractants CCL3-5, CCL8 and CXCL9-11, and did not up-regulate the adhesion molecules ICAM1 and VCAM1. Lack of IFNAR signaling on T lymphocytes impaired Ly-6C expression and lymphocyte function-associated antigen-1 (LFA-1) clustering, which resulted in reduced cell adhesion. Finally, we show that IFNAR-deficiency inhibited T lymphocyte recruitment to the airways during respiratory syncytial virus (RSV) infection.

Chemokines induce Ca2+ signaling and chemotaxis in dendritic cells (DCs) but the molecular players involved in shaping the changes in intracellular Ca2+ remain to be characterized. Using siRNA and knock-out mice, we show that in addition to IP3-mediated Ca2+ release and store-operated Ca2+ entry the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca2+ release, but not Ca2+ influx in mouse DCs. Consistent with this observation, TRPM2 expression in DCs cells is restricted to endolysosomal vesicles, whereas in neutrophils the channel localizes to the plasma membrane and does not participate in Ca2+ release. While TRPM2 is not involved in DCs maturation, TRPM2-deficient DCs show severely compromised chemotaxis regardless of maturation stage. This is due to perturbed Ca2+ signaling manifested through suppression of specific TRPM2-mediated Ca2+-release events and an additional secondary modification of store-operated Ca2+ entry. DCs deficient in both TRPM2 and inositol-1,4,5-trisphosphate receptor signaling (InsP3R) lose their ability to perform chemotaxis entirely. These findings confirm ADPR as a novel second messenger and highlight TRPM2 as a key player regulating DCs chemotaxis through its function as Ca2+ release channel.

Azole derivatives exert direct anti-inflammatory activity in fungal infections and other cutaneous inflammatory conditions. Inhibition of PMN chemotaxis and leukotriene biosynthesis has been suggested to explain this phenomenon, yet the molecular mechanism of action for these drugs remains unknown. Recently we showed that NAD+ derived metabolites, cADPR and ADPR; regulate PMN chemotaxis by inducing extracellular Ca2+ influx via the ion channel TRPM2. Here we investigated whether azole derivatives inhibit PMN chemotaxis in vitro by altering the production of NAD+-derived metabolites or by blocking plasma membrane channel-conducted Ca2+ influx. First, we demonstrated that econazole and clotrimazole significantly inhibited PMN directionality and speed in an EZ-Taxiscan chemotaxis assay. Interestingly, econazole and clotrimazole, as well as FA, another compound proposed to block the TRPM2 channel, blocked fMLP-induced Ca2+ influx, rendering defective Ca2+ responses comparable to those responses observed in fMLP-stimulated TRPM2 KO PMN. Moreover, we found that pre-treatment of neutrophils with econazole or clotrimazole did not alter NAD+ metabolism, but its effect was by blocking a membrane channel with physiological properties similar to TRPM2. Collectively, these data suggest that compounds, which antagonize TRPM2-mediated Ca2+ signaling pathway, may function as effective inhibitors of PMN recruitment, and will likely prove useful in the treatment of inflammatory diseases.