Impairment of neutrophil functions and homeostasis in COVID-19 patients: association with disease severity.

BACKGROUND: A dysregulated immune response is emerging as a key feature of critical illness in COVID-19. Neutrophils are key components of early innate immunity that, if not tightly regulated, contribute to uncontrolled systemic inflammation. We sought to decipher the role of neutrophil phenotypes, functions, and homeostasis in COVID-19 disease severity and outcome. METHODS: By using flow cytometry, this longitudinal study compares peripheral whole-blood neutrophils from 90 COVID-19 ICU patients with those of 22 SARS-CoV-2-negative patients hospitalized for severe community-acquired pneumonia (CAP) and 38 healthy controls. We also assessed correlations between these phenotypic and functional indicators and markers of endothelial damage as well as disease severity. RESULTS: At ICU admission, the circulating neutrophils of the COVID-19 patients showed continuous basal hyperactivation not seen in CAP patients, associated with higher circulating levels of soluble E- and P-selectin, which reflect platelet and endothelial activation. Furthermore, COVID-19 patients had expanded aged-angiogenic and reverse transmigrated neutrophil subsets-both involved in endothelial dysfunction and vascular inflammation. Simultaneously, COVID-19 patients had significantly lower levels of neutrophil oxidative burst in response to bacterial formyl peptide. Moreover patients dying of COVID-19 had significantly higher expansion of aged-angiogenic neutrophil subset and greater impairment of oxidative burst response than survivors. CONCLUSIONS: These data suggest that neutrophil exhaustion may be involved in the pathogenesis of severe COVID-19 and identify angiogenic neutrophils as a potentially harmful subset involved in fatal outcome.

Neutrophil hyperactivation correlates with Alzheimer's disease progression.

OBJECTIVE: Recent studies have underlined the effect of systemic inflammation on the pathophysiology of Alzheimer's disease (AD). Neutrophils are key components of early innate immunity and contribute to uncontrolled systemic inflammation if not tightly regulated. The aim of our study was to fully characterize human circulating neutrophils at different disease stages in AD. METHODS: We analyzed neutrophil phenotypes and functions in 42 patients with AD (16 with mild cognitive impairment and 26 with dementia), and compared them to 22 age-matched healthy subjects. This study was performed directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. RESULTS: Blood samples from AD patients with dementia revealed neutrophil hyperactivation associated with increased reactive oxygen species production and increased levels of intravascular neutrophil extravascular traps. The homeostasis of circulating neutrophils in these patients also changed: The ratio between the harmful hyperreactive CXCR4high /CD62Llow senescent and the CD16bright /CD62Ldim immunosuppressive neutrophil subsets rose in the later stage of the disease. These abnormalities were greater in fast-decliner than in slow-decliner patients. INTERPRETATION: Our results indicate that the inflammatory properties of circulating neutrophils shift as the percentage of aged neutrophils expands in patients with AD-changes that may play an instrumental role in establishing systemic chronic inflammation. Most important, our data strongly suggest that the neutrophil phenotype may be associated with the rate of cognitive decline and may thus constitute an innovative and prognostic blood biomarker in patients with AD. Ann Neurol 2018;83:387-405.

A polysaccharide virulence factor of a human fungal pathogen induces neutrophil apoptosis via NK cells.

Aspergillus fumigatus is an opportunistic human fungal pathogen that sheds galactosaminogalactan (GG) into the environment. Polymorphonuclear neutrophils (PMNs) and NK cells are both part of the first line of defense against pathogens. We recently reported that GG induces PMN apoptosis. In this study, we show that PMN apoptosis occurs via a new NK cell-dependent mechanism. Reactive oxygen species, induced by the presence of GG, play an indispensable role in this apoptotic effect by increasing MHC class I chain-related molecule A expression at the PMN surface. This increased expression enables interaction between MHC class I chain-related molecule A and NKG2D, leading to NK cell activation, which in turn generates a Fas-dependent apoptosis-promoting signal in PMNs. Taken together, our results demonstrate that the crosstalk between PMNs and NK cells is essential to GG-induced PMN apoptosis. NK cells might thus play a role in the induction of PMN apoptosis in situations such as unexplained neutropenia or autoimmune diseases.

Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.

Neutrophils in antiretroviral therapy-controlled HIV demonstrate hyperactivation associated with a specific IL-17/IL-22 environment.

BACKGROUND: Despite control of HIV infection under antiretroviral therapy (ART), immune T-cell activation persists in patients with controlled HIV infection, who are at higher risk of inflammatory diseases than the general population. PMNs play a key role in host defenses against invading microorganisms but also potentiate inflammatory reactions in cases of excessive or misdirected responses. OBJECTIVE: The aim of our study was to analyze PMN functions in 60 ART-treated and controlled HIV-infected patients (viral load, <20 RNA copies/mL; CD4 count, ≥ 350 cells/mm(3)) with (HIV[I] group) and without (HIV[NI] group) diseases related to an inflammatory process and to compare them with 22 healthy control subjects. METHODS: Flow cytometry was used to evaluate PMN functions in whole-blood conditions. We studied in parallel the activation markers of T lymphocytes and monocytes and the proinflammatory cytokine environment. RESULTS: Blood samples from HIV-infected patients revealed basal PMN hyperactivation associated with deregulation of the apoptosis/necrosis equilibrium. Interestingly, this hyperactivation was greater in HIV(I) than HIV(NI) patients and contrasted with a lack of monocyte activation in both groups. The percentage of circulating cells producing IL-17 was also significantly higher in HIV-infected patients than in control subjects and was positively correlated with markers of basal PMN activation. In addition, the detection of IL-22 overproduction in HIV(NI) patients suggests that it might contribute to counteracting chronic inflammatory processes during HIV infection. CONCLUSIONS: This study thus demonstrates the presence of highly activated PMNs in HIV-infected patients receiving effective ART and the association of these cells with a specific IL-17/IL-22 environment.

The protein phosphatase PhzA of A. fumigatus is involved in oxidative stress tolerance and fungal virulence.

Protein phosphatases Z that are unique to the fungal kingdom have been associated to resistance to high salt concentration, cell wall integrity, cell cycle regulation, and oxidative stress in fungi. In Aspergillus fumigatus, it was shown that PHZA is under the control of the transcription factor Skn7 and is only involved in the control of the oxidative stress. Accordingly, the ΔphzA mutant showed a defect in virulence in an experimental model of corneal infection in immunocompetent animals and that the impact on susceptibility to cell wall drugs is only secondary.

HIV/SIV infection primes monocytes and dendritic cells for apoptosis.

Subversion or exacerbation of antigen-presenting cells (APC) death modulates host/pathogen equilibrium. We demonstrated during in vitro differentiation of monocyte-derived macrophages and monocyte-derived dendritic cells (DCs) that HIV sensitizes the cells to undergo apoptosis in response to TRAIL and FasL, respectively. In addition, we found that HIV-1 increased the levels of pro-apoptotic Bax and Bak molecules and decreased the levels of anti-apoptotic Mcl-1 and FLIP proteins. To assess the relevance of these observations in the context of an experimental model of HIV infection, we investigated the death of APC during pathogenic SIV-infection in rhesus macaques (RMs). We demonstrated increased apoptosis, during the acute phase, of both peripheral blood DCs and monocytes (CD14(+)) from SIV(+)RMs, associated with a dysregulation in the balance of pro- and anti-apoptotic molecules. Caspase-inhibitor and death receptors antagonists prevented apoptosis of APCs from SIV(+)RMs. Furthermore, increased levels of FasL in the sera of pathogenic SIV(+)RMs were detected, compared to non-pathogenic SIV infection of African green monkey. We suggest that inappropriate apoptosis of antigen-presenting cells may contribute to dysregulation of cellular immunity early in the process of HIV/SIV infection.

Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus.

A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.

Alveolar fibrocyte percentage is an independent predictor of poor outcome in patients with acute lung injury.

OBJECTIVE: Fibrocytes are mesenchymal progenitors involved in normal and pathologic repair. The aims of this study were: 1) to quantify fibrocytes in bronchoalveolar lavage fluid from patients with or without acute lung injury and acute respiratory distress syndrome; and 2) to evaluate the prognostic value of bronchoalveolar lavage fibrocyte percentage in patients with acute lung injury and acute respiratory distress syndrome. DESIGN: Prospective cohort study. SETTING: Three intensive care units of a large tertiary referral center. PATIENTS: One hundred twenty-two ventilated patients requiring bronchoalveolar lavage were enrolled (62 acute respiratory distress syndrome, 30 acute lung injury, 30-ventilated patients without acute lung injury and acute respiratory distress syndrome). INTERVENTIONS: After bronchoalveolar lavage collection during standard care, the patients were followed up for 28 days and clinical outcome was recorded. Fibrocytes (CD45+/collagen 1+) were quantified in bronchoalveolar lavage by flow cytometry. Comparison of bronchoalveolar lavage fibrocyte percentage from patients with or without acute lung injury and acute respiratory distress syndrome was performed using a Wilcoxon test. A multivariate analysis using a Cox model was performed to study the independent predictors of survival. MEASUREMENTS AND MAIN RESULTS: Fibrocytes were detected in 90 of 92 (98%) bronchoalveolar lavages from patients with acute lung injury and acute respiratory distress syndrome. The median percentage of bronchoalveolar lavage fibrocytes was significantly higher in patients with acute lung injury and acute respiratory distress syndrome (5.0%) in comparison with ventilated control subjects (0.9%, p < .0001). After adjustment for age, comorbidity of malignancy, and severity of illness, a bronchoalveolar lavage fibrocyte percentage >6% was independently associated with a higher 28-day mortality in patients with acute lung injury and acute respiratory distress syndrome (hazard ratio [95% confidence interval] 6.15 [2.78-13.64], p ≤ .0001). Addition of bronchoalveolar lavage fibrocyte percentage in a clinical model predicting mortality in patients with acute lung injury and acute respiratory distress syndrome improved global fit and discriminatory capacity (c-statistic, 0.78-0.85; p = .007). CONCLUSIONS: Fibrocytes are detectable in bronchoalveolar lavage during acute lung injury and acute respiratory distress syndrome. A bronchoalveolar lavage fibrocyte percentage >6% provides an additive prognostic value to clinical predictors and may be useful to identify patients with acute lung injury and acute respiratory distress syndrome at highest risk of an adverse outcome.

AIDS progression is associated with the emergence of IL-17-producing cells early after simian immunodeficiency virus infection.

IL-17 is a potent effector cytokine involved in inflammatory response and antimicrobial defense. We report that SIV infection of rhesus macaques (RMs) results in the emergence of IL-17-expressing cells during the acute phase. This subpopulation appears at day 14 postinfection concomitantly with an increase in TGF-beta and IL-18 expression. This subset, which exhibits phenotypic markers of NK T cells (NKT), rather than Th17 CD4 cells, persists during the chronic phase and is higher in noncontrollers SIV-infected RMs compared with controllers SIV-infected RMs. In contrast, in the nonpathogenic model of SIVagm infection of African green monkeys, no change in the level of IL-17-expressing cells is observed in lymphoid organs. Consistent with the emergence of TGF-beta and IL-18 during the acute phase in SIV-infected RMs, but not in SIV-infected African green monkeys, we demonstrate that in vitro TGF-beta and IL-18 induce the differentiation and expansion of IL-17+NKT+. Altogether, these results demonstrate that IL-17-producing NKT are associated with the pathogenesis of SIV in RMs and suggest that TGF-beta and IL-18 play a role in their development.

Nonpathogenesis of simian immunodeficiency virus infection is associated with reduced inflammation and recruitment of plasmacytoid dendritic cells to lymph nodes, not to lack of an interferon type I response, during the acute phase.

Divergent Toll-like receptor 7 (TLR7) and TLR9 signaling has been proposed to distinguish pathogenic from nonpathogenic simian immunodeficiency virus infection in primate models. We demonstrate here that increased expression of type I interferon in pathogenic rhesus macaques compared to nonpathogenic African green monkeys was associated with the recruitment of plasmacytoid dendritic cells in the lymph nodes and the presence of an inflammatory environment early after infection, instead of a difference in the TLR7/9 response.

Early divergence in neutrophil apoptosis between pathogenic and nonpathogenic simian immunodeficiency virus infections of nonhuman primates.

We used pathogenic and nonpathogenic simian models of SIV infection of Chinese and Indian rhesus macaque (RMs) and African green monkeys (AGMs), respectively, to investigate the relationship between polymorphonuclear neutrophil (PMN) death and the extent of viral replication and disease outcome. In this study, we showed that PMN death increased early during the acute phase of SIV infection in Chinese RMs and coincided with the peak of viral replication on day 14. The level of PMN death was significantly more severe in RMs that progressed more rapidly to AIDS and coincided with neutropenia. Neutropenia was also observed in Indian RMs and was higher in non-Mamu-A*01 compared with Mamu-A*01 animals. In stark contrast, no changes in the levels of PMN death were observed in the nonpathogenic model of SIVagm-sab (sabaeus) infection of AGMs despite similarly high viral replication. PMN death was a Bax and Bak-independent mitochondrial insult, which is prevented by inhibiting calpain activation but not caspases. We found that BOB/GPR15, a SIV coreceptor, is expressed on the PMN surface of RMs at a much higher levels than AGMs and its ligation induced PMN death, suggesting that SIV particle binding to the cell surface is sufficient to induce PMN death. Taken together, our results suggest that species-specific differences in BOB/GPR15 receptor expression on PMN can lead to increased acute phase PMN death. This may account for the decline in PMN numbers that occurs during primary SIV infection in pathogenic SIV infection and may have important implications for subsequent viral replication and disease progression.

Author Correction: Tissue damage from neutrophil-induced oxidative stress in COVID-19.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased neutrophil apoptosis in chronically SIV-infected macaques.

Polymorphonuclear neutrophils (PMN) from chronically HIV-infected individuals have been reported to be more prone to die. However, although non-human primates models have been extensively used for improving our knowledge on T cell immunity, the impact of SIV-infection on PMN, in relationships with disease severity, has never been assessed. In our study, we demonstrate that PMN from Rhesus macaques (RMs) of Chinese origin chronically infected with the virulent strain SIVmac251 display increased susceptibility to undergo apoptosis as compared to PMN from RMs infected with the non-pathogenic SIVDeltanef strain. PMN apoptosis was significantly increased in RMs progressing faster to AIDS as compared to non-progressors RMs. Furthermore, the percentage of apoptotic cells correlated with PMN activation state reflected by increased CD11b expression and reactive oxygen species production. Interestingly, whereas inflammatory cytokines IL-8 and IL-1beta prevent in vitro PMN death, the levels of those cytokines were low in RMs progressing towards AIDS. Altogether, increased PMN death during SIV infection is a new pathogenic effect associated with AIDS progression, adding to the long list of markers associated with disruption of defense against infection.

First description of NOD2 variant associated with defective neutrophil responses in a woman with granulomatous mastitis related to corynebacteria.

We report the first case of granulomatous mastitis due to Corynebacterium kroppenstedtii linked to strongly impaired neutrophil responses to Nod2 agonist and a single nucleotide polymorphism within the NOD2 gene (SNP13 [Leu1007fsinsC]) in a heterozygous state. These findings provided the first demonstration of impaired Nod2 function associated with corynebacterial infection.

Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations.

Flow cytometric analysis provides a rapid screen for abnormalities of polymorphonuclear neutrophils (PMN) function and reflect their behavior in vivo more accurately. This review summarizes the major fluorescent probes used to study PMN oxidative burst and apoptosis using flow cytometry (FCM). We also provide examples of FCM studies in physiological and pathological situations, illustrating the advantages of FCM for assessment of PMN oxidative burst and PMN apoptosis. These data point to the role of FCM in detecting primary immunodeficiencies such as IRAK4 deficiency and support the use of the assessment of the PMN oxidative burst for routine testing in patients with bacterial infections. We also demonstrate the utility of whole-blood analysis using FCM for a better understanding of PMN functionality, i.e., tuning PMN responses to inflammatory stimuli. Finally, FCM permits a simultaneous analysis of phenotypic, functional and morphometric parameters assessing whole-blood PMN apoptosis, in particular in response to Toll-like receptor agonists and during simian immunodeficiency virus infection.

Risks Related to the Use of Non-Steroidal Anti-Inflammatory Drugs in Community-Acquired Pneumonia in Adult and Pediatric Patients.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used to alleviate symptoms during community-acquired pneumonia (CAP), while neither clinical data nor guidelines encourage this use. Experimental data suggest that NSAIDs impair neutrophil intrinsic functions, their recruitment to the inflammatory site, and the resolution of inflammatory processes after acute pulmonary bacterial challenge. During CAP, numerous observational data collected in hospitalized children, hospitalized adults, and adults admitted to intensive care units (ICUs) support a strong association between pre-hospital NSAID exposure and a delayed hospital referral, a delayed administration of antibiotic therapy, and the occurrence of pleuropulmonary complications, even in the only study that has accounted for a protopathic bias. Other endpoints have been described including a longer duration of antibiotic therapy and a greater hospital length of stay. In all adult series, patients exposed to NSAIDs were younger and had fewer comorbidities. The mechanisms by which NSAID use would entail a complicated course in pneumonia still remain uncertain. The temporal hypothesis and the immunological hypothesis are the two main emerging hypotheses. Current data strongly support an association between NSAID intake during the outpatient treatment of CAP and a complicated course. This should encourage experts and scientific societies to strongly advise against the use of NSAIDs in the management of lower respiratory tract infections.

Harmful neutrophil subsets in patients with ischemic stroke: Association with disease severity.

Objective: To better understand the functional state of circulating neutrophils in patients with ischemic stroke (IS) for planning future clinical trials. Methods: We analyzed by flow cytometry activation state of circulating neutrophils and the distribution of neutrophil peripheral subsets in 41 patients with acute IS less than 6 hours before admission and compared them with 22 age-matched healthy controls. Results: Our results demonstrated continuous basal hyperactivation of circulating neutrophils during acute IS, characterized by lower l-selectin expression and higher CD11b expression at the cell surface, increased ROS production by neutrophils, and greater circulating levels of neutrophil elastase. Neutrophil hyperactivation was associated with deregulation of the equilibrium between apoptotic and necrotic. Patients also had higher percentages than controls of the overactive senescent (CXCR4bright/CD62Ldim) neutrophil subset and increased percentage of neutrophils with a reverse transendothelial migration (CD54highCXCR1low) phenotype. Importantly, neutrophil alterations were associated with the clinical severity of the stroke, evaluated by its NIH Stroke Scale score. Conclusion: Altogether, our results indicate that during acute IS, the inflammatory properties of circulating neutrophils rise, associated with the expansion of harmful neutrophil subsets. These changes in neutrophil homeostasis, associated with disease severity, may play an instrumental role by contributing to systemic inflammation and to the blood-brain barrier breakdown. Our findings highlight new potential therapeutic approaches of stroke by rebalancing the ratio of senescent to immunosuppressive neutrophils or decreasing reverse neutrophil transmigration or both.

Molecular epidemiology of chronic granulomatous disease in a series of 80 kindreds: identification of 31 novel mutations.

Chronic granulomatous disease (CGD) results from constitutional inactivating mutations in the CYBB, NCF1, CYBA or NCF2 genes that encode subunits of phagocyte NADPH oxidase. We report the findings of molecular analysis of 80 kindred. In 75 unrelated male and 5 female probands, CGD was suspected on the basis of clinical symptoms, and biological samples were referred to our laboratory between 2000 and 2007. Seventy seven patients were found to have mutations in CYBB, NCF1, CYBA or NCF2 (52 different mutations including 31 mutations not previously reported). CYBB was the most frequently mutated gene (58 males and 3 females, 76%). In autosomal recessive forms of the disease, mutations were found in NCF1 (11 patients), NCF2 (3 patients) and CYBA (2 patients). We observed that significantly fewer females were affected by autosomal recessive CGD than expected (2 females/14 males; p=0.002), suggesting that female patients with CGD may be under diagnosed.

Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity.

Interleukin-10 (IL-10) exerts its anti-inflammatory properties by down-regulating polymorphonuclear neutrophil (PMN) functions such as reactive oxygen species (ROS) production via NADPH oxidase. The molecular mechanisms underlying this process are unclear. Partial phosphorylation of the NADPH oxidase cytosolic component p47(PHOX) induced by proinflammatory cytokines, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha, is essential for priming ROS production by PMN. The aim of this study was to determine whether IL-10 inhibits GM-CSF- and TNFalpha-induced p47(PHOX) phosphorylation and to investigate the molecular mechanisms involved in this effect. We found that IL-10 selectively inhibited GM-CSF- but not TNFalpha-induced p47PHOX phosphorylation in a concentration-dependent manner. As GM-CSF-induced p47PHOX phosphorylation is mediated by extracellular signal-regulated kinase 1/2 (ERK1/2), we tested the effect of IL-10 on this pathway. We found that IL-10 inhibited GM-CSF-induced ERK1/2 activity in an immunocomplex kinase assay. This inhibitory effect was confirmed by analyzing the phosphorylation status of the endogenous substrate of ERK1/2, p90RSK, in intact PMN. Furthermore, IL-10 decreased ROS production by adherent GM-CSF-treated PMN in keeping with the higher ROS production observed in whole blood from IL-10 knockout mice compared to their wild-type counterparts. Together, these results suggest that IL-10 inhibits GM-CSF-induced priming of ROS production by inhibiting p47PHOX phosphorylation through a decrease in ERK1/2 activity. This IL-10 effect could contribute to the tight regulation of NADPH oxidase activity at the inflammatory site.