Reactive oxygen species (ROS) produced by NADPH phagocyte oxidase isoform (NOX2) are critical for the elimination of intracellular pathogens in many infections. Despite their importance, the role of ...ROS following infection with the eukaryotic pathogen
has not been fully elucidated. We addressed the role of ROS in C57BL/6 mice following intradermal infection with
Despite equivalent parasite loads compared with wild-type (WT) mice, mice deficient in ROS production by NOX2 due to the absence of the gp91 subunit (gp91
) had significantly more severe pathology in the later stages of infection. Pathology in gp91
mice was not associated with alterations in CD4
T cell-mediated immunity but was preceded by enhanced neutrophil accumulation at the dermal infection site. Ex vivo analysis of infected versus uninfected neutrophils revealed a deficiency in infection-driven apoptosis in gp91
mice versus WT mice. gp91
mice presented with higher percentages of healthy or necrotic neutrophils but lower percentages of apoptotic neutrophils at early and chronic time points. In vitro infection of gp91
versus WT neutrophils also revealed reduced apoptosis and CD95 expression but increased necrosis in infected cells at 10 h postinfection. Provision of exogenous ROS in the form of H
O
reversed the necrotic phenotype and restored CD95 expression on infected gp91
neutrophils. Although ROS production is typically viewed as a proinflammatory event, our observations identify the importance of ROS in mediating appropriate neutrophil apoptosis and the importance of apoptosis in inflammation and pathology during chronic infection.
The route of pathogen inoculation by needle has been shown to influence the outcome of infection. Employing needle inoculation of the obligately intracellular parasite Leishmania major, which is ...transmitted in nature following intradermal (i.d.) deposition of parasites by the bite of an infected sand fly, we identified differences in the preexisting and acute cellular responses in mice following i.d. inoculation of the ear, subcutaneous (s.c.) inoculation of the footpad, or inoculation of the peritoneal cavity (intraperitoneal i.p. inoculation). Initiation of infection at different sites was associated with different phagocytic populations. Neutrophils were the dominant infected cells following i.d., but not s.c. or i.p., inoculation. Inoculation of the ear dermis resulted in higher frequencies of total and infected neutrophils than inoculation of the footpad, and these higher frequencies were associated with a 10-fold increase in early parasite loads. Following inoculation of the ear in the absence of neutrophils, parasite phagocytosis by other cell types did not increase, and fewer parasites were able to establish infection. The frequency of infected neutrophils within the total infected CD11b(+) population was higher than the frequency of total neutrophils within the total CD11b(+) population, demonstrating that neutrophils are overrepresented as a proportion of infected cells. Employing i.d. inoculation to model sand fly transmission of parasites has significant consequences for infection outcome relative to that of s.c. or i.p. inoculation, including the phenotype of infected cells and the number of parasites that establish infection. Vector-borne infections initiated in the dermis likely involve adaptations to this unique microenvironment. Bypassing or altering this initial step has significant consequences for infection.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in controlling several aspects of immune responses, including the activation and differentiation of specific T ...cell subsets and antigen-presenting cells, thought to be relevant in the context of experimental Trypanosoma cruzi infection. The relevance of AhR for the outcome of T. cruzi infection is not known and was investigated here. We infected wild-type (WT) mice and AhR knockout (AhR KO) mice with T. cruzi (Y strain) and determined levels of parasitemia, myocardial inflammation and fibrosis, expression of AhR/cytokines/suppressor of cytokine signaling (SOCS) (spleen/heart), and production of nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (ONOO(-)) (spleen). AhR expression was increased in the heart of infected WT mice. Infected AhR KO mice displayed significantly reduced parasitemia, inflammation, and fibrosis of the myocardium. This was associated with an anticipated increased immune response characterized by increased levels of inflammatory cytokines and reduced expression of SOCS2 and SOCS3 in the heart. In vitro, AhR deficiency caused impairment in parasite replication and decreased levels of ROS production. In conclusion, AhR influences the development of murine Chagas disease by modulating ROS production and regulating the expression of key physiological regulators of inflammation, SOCS1 to -3, associated with the production of cytokines during experimental T. cruzi infection.
The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional ...epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease–susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress–ATF6–DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.
•IL-18 contributes to susceptibility to L. amazonensis infection in C57BL/6 mice.•Macrophages do not contribute to these effects of IL-18.•BMMϕ do not express IL-18R constitutively or under any of ...the conditions studied.
We evaluated the role of IL-18 during Leishmania amazonensis infection in C57BL/6 mice, using IL-18KO mice. We showed that IL-18 is involved in susceptibility to L. amazonensis, since IL-18KO mice presented reduced lesions and parasite loads. Because macrophages are the host cells of the parasite, we investigated if macrophages were involved in IL-18-mediated susceptibility to L. amazonensis. We showed that macrophages obtained from WT or IL-18KO responded similarly to L. amazonensis infection. Moreover, we showed that C57BL/6 macrophages do not respond to IL-18, since they do not express IL-18R. Therefore, macrophages are not involved in IL-18-mediated susceptibility to L. amazonensis.
C57BL/6 mice macrophages innately produce higher levels of NO than BALB/c cells when stimulated with LPS. Here, we investigated the molecular events that account for this intrinsic differential ...production of NO. We found that the lower production of NO in BALB/c is not due to a subtraction of L-arginine by arginase, and correlates with a lower iNOS accumulation, which is independent of its degradation rate. Instead, the lower accumulation of iNOS is due to the lower levels of iNOS mRNA, previously shown to be also independent of its stability, suggesting that iNOS transcription is less efficient in BALB/c than in C57BL/6 macrophages. Activation of NFκB is more efficient in BALB/c, thus not correlating with iNOS expression. Conversely, activation of STAT-1 does correlate with iNOS expression, being more prominent in C57BL/6 than in BALB/c macrophages. IFN-β and IL-10 are more highly expressed in C57BL/6 than in BALB/c macrophages, and the opposite is true for TNF-α. Whereas IL-10 and TNF-α do not seem to participate in their differential production of NO, IFN-β has a determinant role since 1) anti-IFN-β neutralizing antibodies abolish STAT-1 activation reducing NO production in C57BL/6 macrophages to levels as low as in BALB/c cells and 2) exogenous rIFN-β confers to LPS-stimulated BALB/c macrophages the ability to phosphorylate STAT-1 and to produce NO as efficiently as C57BL/6 cells. We demonstrate, for the first time, that BALB/c macrophages are innately lower NO producers than C57BL/6 cells because they are defective in the TLR-4-induced IFN-β-mediated STAT-1 activation pathway.
Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen ...we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-γ dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-γ dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen.
Abstract
The protozoan Leishmania parasite is a human obligate intra-cellular pathogen and the causative agent of leishmaniasis, a chronic neglected tropical disease of existing and growing global ...importance. Despite considerable effort a human vaccine does not exist. In the C57Bl/6 mouse model of naturally acquired protective immunity, chronic primary infection maintains short-lived Ly6C+ TEFF cells that mediate protective immunity against secondary challenge. These cells, not memory cells or cells derived from memory cells following challenge, emulate the rapid IFN-g response associated with protective immunity against natural infected sand fly challenge. Defining the mechanisms by which these cells mediate their protective effect will be essential for informing future vaccine design. To elucidate the importance of the rapidity of the Ly6C-mediated response as a protective correlate, the influence of these cells on innate immune cells, and ultimately their capacity to facilitate the priming and activation of Leishmania-specific naïve cells, we devised an adoptive transfer system in which Ly6C+ TEFF cells from chronic mice are transferred into recipient mice at the time of infection or 4 days following infection, just prior to the initiation of parasite replication. Disease progression was followed for 3 weeks and the activation of phagocytes, antigen-presenting cell function, and IFN-g production by both adoptively transferred and recipient populations was analyzed. Our observations are likely to enhance our understanding of the correlates of protective immunity beyond the simple generation of IFN-g producing memory cells, a strategy that has so far failed to generate an effective human vaccine.
Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen ...we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-gamma dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-gamma dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen.
The main purpose of the investigation reported here was to analyze the effect of resistance training (RT) performed at different weekly frequencies on flexibility in older women.
Fifty-three older ...women (≥60 years old) were randomly assigned to perform RT either two (n=28; group "G2x"), or three (n=25; group "G3x") times per week. The RT program comprised eight exercises in which the participants performed one set of 10-15 repetitions maximum for a period of 12 weeks. Anthropometric, body-composition, and flexibility measurements were made at baseline and post-study. The flexibility measurements were obtained by a fleximeter.
A significant group-by-time interaction (P<0.01) was observed for frontal hip flexion, in which G3x showed a higher increase than G2x (+12.8% and +3.0%, respectively). Both groups increased flexibility in cervical extension (G2x=+19.1%, G3x=+20.0%), right hip flexion (G2x=+14.6%, G3x=+15.9%), and left hip flexion (G2x=+25.7%, G3x=+19.2%), with no statistical difference between groups. No statistically significant differences were noted for the increase in skeletal muscle mass between training three versus two times a week (+7.4% vs +4.4%, respectively).
Twelve weeks of RT improves the flexibility of different joint movements in older women, and the higher frequency induces greater increases for frontal hip flexion.
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