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Severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS–CoV-2 infections and that CASP4 expression correlates with severity of SARS–CoV-2 infection in humans. SARS–CoV-2–infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS–CoV-2–infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1β, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS–CoV-2–induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

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.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Herein, we describe an extracellular function of the vertebrate high-mobility group box 1 protein (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 functions as a DNA architectural protein, similar to the ubiquitous DNABII family of bacterial proteins; despite that, these proteins share no amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, whereas the DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. We showed that when both proteins converged on extracellular DNA within bacterial biofilms, HMGB1, unlike the DNABII proteins, disrupted biofilms both in vitro (including the high-priority ESKAPEE pathogens) and in vivo in 2 distinct animal models, albeit with induction of a strong inflammatory response that we attenuated by a single engineered amino acid change. We propose a model where extracellular HMGB1 balances the degree of induced inflammation and biofilm containment without excessive release of biofilm-resident bacteria.

Aim: To characterize early events in neotissue formation during the first 2 weeks after vascular scaffold implantation. Materials & methods: Biodegradable polymeric scaffolds were implanted as abdominal inferior vena cava interposition grafts in wild-type mice. Results: All scaffolds explanted at day 1 contained a platelet-rich mural thrombus. Within the first few days, the majority of cell infiltration appeared to be from myeloid cells at the peritoneal surface with modest infiltration along the lumen. Host reaction to the graft was distinct between the scaffold and mural thrombus; the scaffold stimulated an escalating foreign body reaction, whereas the thrombus was quickly remodeled into collagen-rich neotissue. Conclusion: Mural thrombi remodel into neotissue that persistently occludes the lumen of vascular grafts.

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.

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.

Abdominal aortic aneurysm (AAA) is characterized by transmural infiltration of myeloid cells at the vascular injury site. Previously, we reported preventive effects of Notch deficiency on the development of AAA by reduction of infiltrating myeloid cells. In this study, we examined if Notch inhibition attenuates the progression of pre-established AAA and potential implications. Pharmacological Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPT) was administered subcutaneously three times a week starting at day 28 of angiotensin II (AngII) infusion. Progressive increase in pulse wave velocity (PWV), maximal intra-luminal diameter (MILD) and maximal external aortic diameter (MEAD) were observed at day 56 of the AngII. DAPT prevented such increase in MILD, PWV and MEAD (P Apoe−/− mice had significant reduction in inflammatory response and elastin fragmentation. Naked collagen microfibrils and weaker banded structure observed in the aortae of Apoe−/− mice in response to AngII, were substantially diminished by DAPT. A significant decrease in the proteolytic activity in the aneurysmal tissues and vascular smooth muscle cells (vSMCs) was observed with DAPT (P 2 = 0.61). Collectively, we propose inhibition of Notch signaling as a potential therapeutic target for AAA progression.

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.

The liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migration in vitro and in vivo. Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR−/−) LDLR−/− mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

Supplemental Digital Content is available in the text., Objective— Despite successful translation of bioresorbable vascular grafts for the repair of congenital heart disease, stenosis remains the primary cause of graft failure. In this study, we investigated the efficacy of long-term treatment with the antiplatelet drugs, aspirin and cilostazol, in preventing stenosis and evaluated the effect of these drugs on the acute phase of inflammation and tissue remodeling. Approach and Results— C57BL/6 mice were fed a drug-mixed diet of aspirin, cilostazol, or normal chow during the course of follow-up. Bioresorbable vascular grafts, composed of poly(glycolic acid) mesh sealed with poly(l-lactide-co-ε-caprolactone), were implanted as inferior vena cava interposition conduits and followed up for 2 weeks (n=10 per group) or 24 weeks (n=15 per group). Both aspirin and cilostazol suppressed platelet activation and attachment onto the grafts. On explant at 24 weeks, well-organized neotissue had developed, and cilostazol treatment resulted in 100% graft patency followed by the aspirin (67%) and no-treatment (60%) groups (P

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.

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo . Here we identified ribonuclease 6 (RNase 6) as the RNase A superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are upregulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14 + monocytes and murine bone marrow–derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

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.

Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment, and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days postinfection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathological features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.

Chemokines induce calcium (Ca2+) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca2+ changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release and store-operated Ca2+ entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca2+release but not Ca2+ influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca2+ signaling mechanisms, which include suppression of TRPM2-mediated Ca2+ release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP3 receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca2+ release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca2+ mobilization.—Sumoza-Toledo, A., Lange, I., Cortado, H., Bhagat, H., Mori, Y., Fleig, A., Penner, R., Partida-Sánchez, S. Dendritic cell maturation and chemotaxis is regulated by TRPM2-mediated lysosomal Ca2+ release.

Calcium signaling in phagocytes is essential for cellular activation, migration, and the potential resolution of infection or inflammation. The generation of reactive oxygen species (ROS) via activation of NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase activity in macrophages has been linked to altered intracellular calcium concentrations. Because of its role as an oxidative stress sensor in phagocytes, we investigated the function of the cation channel transient receptor potential melastatin 2 (TRPM2) in macrophages during oxidative stress responses induced by Helicobacter pylori infection. We show that Trpm2-/- mice, when chronically infected with H. pylori, exhibit increased gastric inflammation and decreased bacterial colonization compared with wild-type (WT) mice. The absence of TRPM2 triggers greater macrophage production of inflammatory mediators and promotes classically activated macrophage M1 polarization in response to H. pylori. TRPM2-deficient macrophages upon H. pylori stimulation are unable to control intracellular calcium levels, which results in calcium overloading. Furthermore, increased intracellular calcium in TRPM2-/- macrophages enhanced mitogen-activated protein kinase and NADPH-oxidase activities, compared with WT macrophages. Our data suggest that augmented production of ROS and inflammatory cytokines with TRPM2 deletion regulates oxidative stress in macrophages and consequently decreases H. pylori gastric colonization while increasing inflammation in the gastric mucosa.

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.

CD38 controls the chemotaxis of leukocytes to some, but not all, chemokines, suggesting that chemokine receptor signaling in leukocytes is more diverse than previously appreciated. To determine the basis for this signaling heterogeneity, we examined the chemokine receptors that signal in a CD38-dependent manner and identified a novel "alternative" chemokine receptor signaling pathway. Similar to the "classical" signaling pathway, the alternative chemokine receptor pathway is activated by Galpha(i2)-containing Gi proteins. However, unlike the classical pathway, the alternative pathway is also dependent on the Gq class of G proteins. We show that Galpha(q)-deficient neutrophils and dendritic cells (DCs) make defective calcium and chemotactic responses upon stimulation with N-formyl methionyl leucyl phenylalanine and CC chemokine ligand (CCL) 3 (neutrophils), or upon stimulation with CCL2, CCL19, CCL21, and CXC chemokine ligand (CXCL) 12 (DCs). In contrast, Galpha(q)-deficient T cell responses to CXCL12 and CCL19 remain intact. Thus, the alternative chemokine receptor pathway controls the migration of only a subset of cells. Regardless, the novel alternative chemokine receptor signaling pathway appears to be critically important for the initiation of inflammatory responses, as Galpha(q) is required for the migration of DCs from the skin to draining lymph nodes after fluorescein isothiocyanate sensitization and the emigration of monocytes from the bone marrow into inflamed skin after contact sensitization.

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. Here we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C (PKC) expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production following PMA stimulation compared to non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent PKC conventional subclass α/β leading to reduced phosphorylation of NADPH oxidase components p47phox & p40phox in comparison to non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared to non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst due to 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 in order to limit infection.

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages.

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.

Migration of dendritic cells (DCs) to the draining lymph node (DLN) is required for the activation of naive T cells. We show here that migration of DCs from the lung to the DLN after Mycobacterium tuberculosis (Mtb) exposure is defective in mice lacking interleukin (IL)-12p40. This defect compromises the ability of IL-12p40–deficient DCs to activate naive T cells in vivo; however, DCs that express IL-12p40 alone can activate naive T cells. Treatment of IL-12p40–deficient DCs with IL-12p40 homodimer (IL-12(p40)2) restores Mtb-induced DC migration and the ability of IL-12p40–deficient DCs to activate naive T cells. These data define a novel and fundamental role for IL-12p40 in the pathogen-induced activation of pulmonary DCs.

Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.

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.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

A rat monoclonal antibody (mAb) (NIM-R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes ('spreading') in B lymphocytes activated by anti-IgM and interleukin-4 (IL-4) or anti-CD38 and IL-4. In contrast, resting B cells were unable to spread similarly on the NIM-R8-coated plates. The NIM-R8 antibody recognized a 90,000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccharide, anti-IgM plus IL-4 or concanavalin A) activation. The NIM-R8 mAb with or without IL-2 or IL-4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM-R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand-inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell-surface area for cell-cell or cell-matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes.

Polyriboinosinic-polyribocytoidylic acid (pIC), a synthetic dsRNA, acts as an adjuvant that boosts immune responses and protection. Intranasal (IN) administration of pIC has recently been used to adjuvant influenza virus vaccines; however, the effects of IN pIC administration on pulmonary T cell responses remain unclear. Here we show that a single IN administered dose of dsRNA into mice induced local Th1 chemokine production in the lungs and airways, and generated a biphasic and sustained migration of T lymphocytes to the airways. Furthermore, IN pIC-induced chemokine production and T cell recruitment to the airways were interferon-α receptor (IFNAR) signaling dependent. The effect of dsRNA on T cell recruitment to the airways was also dependent on the presence of high molecular weight (HMW) pIC, as a low molecular weight (LMW) pIC preparation known to only interact with TLR3 did not elicit the same effect on T cell migration to the airways, suggesting that the observed effects were dependent upon dsRNA recognition by multiple pattern recognition receptors (PPRs). IN pIC was additionally capable of stimulating low levels of T cell proliferation in the draining lymph nodes approximately 4-6 days after treatment that preceded a small population of de-novo T cells found in the airways by day 10. Taken together, these results demonstrate that the adjuvant effect of IN pIC that results in enhanced T cell proliferation and sustained T cell recruitment to the airways requires multiple PRRs and IFNAR signaling.

Objective— Activation of inflammatory pathways plays a critical role in the development of abdominal aortic aneurysms (AAA). Notch1 signaling is a significant regulator of the inflammatory response; however, its role in AAA is unknown. Methods and Results— In an angiotensin II–induced mouse model of AAA, activation of Notch1 signaling was observed in the aortic aneurysmal tissue of Apoe −/− mice, and a similar activation of Notch1 was observed in aneurysms of humans undergoing AAA repair. Notch1 haploinsufficiency significantly reduced the incidence of AAA in Apoe −/− mice in response to angiotensin II. Reconstitution of bone marrow–derived cells from Notch1 +/− ;Apoe −/− mice (donor) in lethally irradiated Apoe −/− mice (recipient) decreased the occurrence of aneurysm. Flow cytometry and immunohistochemistry demonstrated that Notch1 haploinsufficiency prevented the influx of inflammatory macrophages at the aneurysmal site by causing defects in macrophage migration and proliferation. In addition, there was an overall reduction in the inflammatory burden in the aorta of the Notch1 +/− ;Apoe −/− mice compared with the Apoe −/− mice. Last, pharmacological inhibition of Notch1 signaling also prevented AAA formation and progression in Apoe −/− mice. Conclusion— Our data suggest that decreased levels of Notch1 protect against the formation of AAA by preventing macrophage recruitment and attenuating the inflammatory response in the aorta.

Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20-80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organs.

Uropathogenic Escherichia coli proceed through a complex intracellular developmental pathway that includes multiple morphological changes. During intracellular growth within Toll-like receptor 4-activated superficial bladder epithelial cells, a subpopulation of uropathogenic E. coli initiates SulA-mediated filamentation. In this study, we directly investigated the role of bacterial morphology in the survival of uropathogenic E. coli from killing by phagocytes. We initially determined that both polymorphonuclear neutrophils and macrophages are recruited to murine bladder epithelium at times coincident with extracellular bacillary and filamentous uropathogenic E. coli. We further determined that bacillary uropathogenic E. coli were preferentially destroyed when mixed uropathogenic E. coli populations were challenged with cultured murine macrophages in vitro. Consistent with studies using elliptical-shaped polymers, the initial point of contact between the phagocyte and filamentous uropathogenic E. coli influenced the efficacy of internalization. These findings demonstrate that filamentous morphology provides a selective advantage for uropathogenic E. coli evasion of killing by phagocytes and defines a mechanism for the essential role for SulA during bacterial cystitis. Thus, morphological plasticity can be viewed as a distinct class of mechanism used by bacterial pathogens to subvert host immunity.

TRPM2 is a Ca 2+ -permeable cation channel that is specifically activated by adenosine diphosphoribose (ADPR). Channel activation in the plasma membrane leads to Ca 2+ influx and has been linked to apoptotic mechanisms. The primary agonist, ADPR, is produced both extra- and intracellularly and causes increases in intracellular calcium concentration ([Ca 2+ ] i ), but the mechanisms involved are not understood. Using short interfering RNA and a knockout mouse, we report that TRPM2, in addition to its role as a plasma membrane channel, also functions as a Ca 2+ -release channel activated by intracellular ADPR in a lysosomal compartment. We show that both functions of TRPM2 are critically linked to hydrogen peroxide–induced β cell death. Additionally, extracellular ADPR production by the ectoenzyme CD38 from its substrates NAD + (nicotinamide adenine dinucleotide) or cADPR causes IP 3 -dependent Ca 2+ release via P2Y and adenosine receptors. Thus, ADPR and TRPM2 represent multimodal signaling elements regulating Ca 2+ mobilization in β cells through membrane depolarization, Ca 2+ influx, and release of Ca 2+ from intracellular stores.

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD+. Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD+ analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.

Through its production of cyclic adenosine diphosphate ribose, the ecto-enzyme CD38 regulates calcium mobilization in neutrophils responding to some, but not all, chemoattractants. This signaling defect results in reduced chemotaxis of CD38-deficient neutrophils to bacterial peptides and increased susceptibility of CD38-deficient mice to bacterial infections.

Lymphotoxin-α−/− (LTα−/−) mice are thought to be unable to generate effective T and B cell responses. This is attributed to the lack of lymph nodes and the disrupted splenic architecture of these mice. However, despite these defects we found that LTα−/− mice could survive infection with a virulent influenza A virus. LTα−/− mice and normal wild-type mice infected with influenza A generated similar numbers of influenza-specific CD8 T cells that were able to produce IFN-γ and kill target cells presenting influenza peptides. Furthermore influenza-infected LTα−/− mice produced high titers of influenza-specific IgM, IgG, and IgA. However, both CD8 and B cell immune responses were delayed in LTα−/− mice by 2–3 days. The delayed cellular and humoral immune response was sufficient to mediate viral clearance in LTα−/− mice that were infected with relatively low doses of influenza virus. However, when LTα−/− mice were infected with larger doses of influenza, they succumbed to infection before the immune response was initiated. These results demonstrate that neither LTα nor constitutively organized lymphoid tissues, such as lymph nodes and spleen, are absolutely required for the generation of effective immunity against the respiratory virus influenza A. However, the presence of LTα and/or lymph nodes does accelerate the initiation of immune responses, which leads to protection from larger doses of virus.

Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.