RESUMEN

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality and delayed clearance of the virus. Generating effective CD8+ T cell responses may be important for improving vaccination outcomes in vulnerable populations, but neonatal T cells frequently respond differently than adult cells. We sought to understand CD8+ T cell specificity and immunodominance during neonatal influenza infection and how any differences from the adult hierarchy might impact peptide vaccine effectiveness. Neonatal C57BL/6 mice displayed an altered CD8+ T cell immunodominance hierarchy during influenza infection, preferentially responding to an epitope in the influenza protein PA rather than the co-dominant adult response to NP and PA. Heterosubtypic infections in mice first infected as pups also displayed altered immunodominance and reduced protection compared to mice first infected as adults. Adoptive transfer of influenza-infected bone-marrow-derived dendritic cells promoted an NP-specific CD8+ T cell response in influenza-virus-infected pups and increased viral clearance. Finally, pups responded to PA (224-233), but not NP (366-374) during peptide vaccination. PA (224-233)-vaccinated mice were not protected during viral challenge. Epitope usage should be considered when designing vaccines that target T cells when the intended patient population includes infants and adults.

Asunto(s)

Animales Recién Nacidos , Linfocitos T CD8-positivos , Epítopos Inmunodominantes , Vacunas contra la Influenza , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae , Animales , Linfocitos T CD8-positivos/inmunología , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Epítopos Inmunodominantes/inmunología , Vacunación , Femenino , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificación , Epítopos de Linfocito T/inmunología

RESUMEN

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

RESUMEN

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

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COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

Asunto(s)

COVID-19 , Aprendizaje Profundo , Humanos , ARN Viral , SARS-CoV-2 , Plaquetas/ultraestructura , Orgánulos

RESUMEN

BACKGROUND: HIV-1 infection is associated with multiple procoagulant changes and increased thrombotic risk. Possible mechanisms for this risk include heigthened expression of procoagulant tissue factor (TF) on circulating monocytes, extracellular vesicles, and viral particles and/or acquired deficiency of protein S (PS), a critical cofactor for the anticoagulant protein C (PC). PS deficiency occurs in up to 76% of people living with HIV-1 (PLWH). As increased ex vivo plasma thrombin generation is a strong predictor of mortality, we investigated whether PS and plasma TF are associated with plasma thrombin generation. METHODS: We analyzed plasma samples from 9 healthy controls, 17 PLWH on first diagnosis (naive), and 13 PLWH on antiretroviral therapy (ART). Plasma thrombin generation, total and free PS, PC, C4b-binding protein, and TF activity were measured. RESULTS: We determined that the plasma thrombin generation assay is insensitive to PS, because of a lack of PC activation, and developed a modified PS-sensitive assay. Total plasma PS was reduced in 58% of the naive and 38% of the ART-treated PLWH samples and correlated with increased thrombin generation in the modified assay. Conversely, plasma TF was not increased in our patient population, suggesting that it does not significantly contribute to ex vivo plasma thrombin generation. CONCLUSION: These data suggest that reduced total plasma PS contributes to the thrombotic risk associated with HIV-1 infection and can serve as a prothrombotic biomarker. In addition, our refined thrombin generation assay offers a more sensitive tool to assess the functional consequences of acquired PS deficiency in PLWH.

Asunto(s)

Infecciones por VIH , Proteína S , Biomarcadores , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Humanos , Trombina/metabolismo , Tromboplastina

RESUMEN

Introduction: Long COVID is the overarching name for a wide variety of disorders that may follow the diagnosis of acute SARS-COVID-19 infection and persist for weeks to many months. Nearly every organ system may be affected. Methods: We report nine patients suffering with Long COVID for 101 to 547 days. All exhibited significant perturbations of their immune systems, but only one was known to be immunodeficient prior to the studies directed at evaluating them for possible treatment. Neurological and cardiac symptoms were most common. Based on this data and other evidence suggesting autoimmune reactivity, we planned to treat them for 3 months with long-term high-dose immunoglobulin therapy. If there was evidence of benefit at 3 months, the regimen was continued. Results: The patients' ages ranged from 34 to 79 years-with five male and four female patients, respectively. All nine patients exhibited significant immune perturbations prior to treatment. One patient declined this treatment, and insurance support was not approved for two others. The other six have been treated, and all have had a significant to remarkable clinical benefit. Conclusion: Long-term high-dose immunoglobulin therapy is an effective therapeutic option for treating patients with Long COVID.

Asunto(s)

COVID-19 , Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , COVID-19/etiología , Síndrome Post Agudo de COVID-19 , Pulmón , Inmunoglobulinas , Inmunización Pasiva/efectos adversos

RESUMEN

Newborn mice are unable to clear Pneumocystis (PC) infection with the same efficiency as adults due, in part, to their inability to develop a robust immune response to infection until three weeks of age. It is known that infants tend develop a Th2 skewed response to antigen so we sought to determine whether a biased cytokine response altered the clearance of PC infection in neonatal mice. P. murina infection in neonatal mice resulted in increased IL-4 expression by CD4 T cells and myeloid cells, augmented IL-13 secretion within the airways and increased arginase activity in the airways, indicative of Th2-type responses. P. murina-infected IL-4Rα-/- neonates had a shift towards Th1 cytokine production and increased numbers of CD4 and CD8 T cells within the lung as well as elevated levels of P. murina-specific IgG. IFNγ-/- and IL-23 p19-/- mice had altered CD4-T cell-dependent cytokine and cell responses. Though we could alter the T helper cell environment in neonatal knockout mice, there was no loss in the ability of these pups to clear infection. It is possible that the Th2 phenotype normally seen in neonatal mice protects the developing lung from pro-inflammatory immune responses without compromising host defense against P. murina.

RESUMEN

Regulatory properties of macrophages associated with alternative activation serve to limit the exaggerated inflammatory response during pneumonia caused by Pseudomonas aeruginosa infection. Arginase-1 is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. We investigated the role of arginase-1 in the control of inflammatory immune responses to P. aeruginosa pneumonia in mice that exhibit different immunologic phenotypes. C57BL/6 mice with conditional knockout of the arginase-1 (Arg1) gene from myeloid cells (Arg1ΔM) or BALB/c mice treated with small molecule inhibitors of arginase were infected intratracheally with P. aeruginosa. Weight loss, mortality, bacterial clearance, and lung injury were assessed and compared, as were the characterization of immune cell populations over time post-infection. Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory responses compared to littermate control mice. Arg1ΔM mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to littermate controls. Additionally, Arg1ΔM mice recovered from inflammatory lung injury at a significantly slower rate. Conversely, treatment of BALB/c mice with the arginase inhibitor S-(2-boronoethyl)-l-cysteine hydrochloride (BEC) did not change morbidity as defined by weight loss, but mice at day 10 post-infection treated with BEC had gained significantly more weight back than controls. Neutrophil and macrophage infiltration were similar between groups in the lung parenchyma, and neutrophil migration into the airways was reduced by BEC treatment. Differences seem to lie in the impact on T cell subset disposition. Arg1ΔM mice had increased total CD4+ T cell expansion in the lymph nodes, and increased T cell activation, IFNγ production, and IL-17 production in the lymph nodes, lung interstitium, and airways, while treatment with BEC had no impact on T cell activation or IL-17 production, but reduced the number of T cells producing IFNγ in the lungs. Lung injury scores were increased in the Arg1ΔM mice, but no differences were observed in the mice treated with pharmacologic arginase inhibitors. Overall, myeloid arginase production was demonstrated to be essential for control of damaging inflammatory responses associated with P. aeruginosa pneumonia in C57BL/6 mice, in contrast to a protective effect in the Th2-dominant BALB/c mice when arginase activity is globally inhibited.

Asunto(s)

Arginasa/metabolismo , Linfocitos T CD4-Positivos/inmunología , Inflamación/inmunología , Pulmón/metabolismo , Neutrófilos/inmunología , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/fisiología , Subgrupos de Linfocitos T/inmunología , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Ácidos Borónicos/farmacología , Citocinas/metabolismo , Femenino , Antecedentes Genéticos , Predisposición Genética a la Enfermedad , Humanos , Inmunomodulación , Pulmón/patología , Activación de Linfocitos , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Balance Th1 - Th2

RESUMEN

OBJECTIVE: Thrombocytopenia is associated with many viral infections suggesting virions interact with and affect platelets. Consistently, viral particles are seen inside platelets, and platelet activation markers are detected in viremic patients. In this article, we sought mechanistic insights into these virion/platelet interactions by examining how platelets endocytose, traffic, and are activated by a model virion. Approach and Results: Using fluorescently tagged HIV-1 pseudovirions, 3-dimensional structured illumination microscopy, and transgenic mouse models, we probed the interactions between platelets and virions. Mouse platelets used known endocytic machinery, that is, dynamin, VAMP (vesicle-associated membrane protein)-3, and Arf6 (ADP-ribosylation factor 6), to take up and traffic HIV-1 pseudovirions. Endocytosed HIV-1 pseudovirions trafficked through early (Rab4+) and late endosomes (Rab7+), and then to an LC3+ (microtubule-associated protein 1A/1B-light chain 3) compartment. Incubation with virions induced IRAK4 (interleukin 1 receptor-associated kinase 4), Akt (protein kinase B), and IKK (IκB kinase) activation, granule secretion, and platelet-leukocyte aggregate formation. This activation required TLRs (Toll-like receptors) and MyD88 (myeloid differentiation primary response protein 88) but was less extensive and slower than activation with thrombin. In vivo, HIV-1 pseudovirions injection led to virion uptake and platelet activation, as measured by IKK activation, platelet-leukocyte aggregate formation, and mild thrombocytopenia. All were decreased in VAMP-3-/- and, megakaryocyte/platelet-specific, Arf6-/- mice. Similar platelet activation profiles (increased platelet-leukocyte aggregates, plasma platelet factor 4, and phospho-IκBα) were detected in newly diagnosed and antiretroviral therapy-controlled HIV-1+ patients. CONCLUSIONS: Collectively, our data provide mechanistic insights into the cell biology of how platelets endocytose and process virions. We propose a mechanism by which platelets sample the circulation and respond to potential pathogens that they take up.

Asunto(s)

Plaquetas/metabolismo , Endocitosis , Infecciones por VIH/sangre , VIH-1/patogenicidad , Activación Plaquetaria , Trombocitopenia/sangre , Receptores Toll-Like/sangre , Virión , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/sangre , Factores de Ribosilacion-ADP/genética , Animales , Antirretrovirales/uso terapéutico , Plaquetas/virología , Agregación Celular , Células Cultivadas , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Quinasa I-kappa B/sangre , Quinasa I-kappa B/genética , Leucocitos/metabolismo , Leucocitos/virología , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/sangre , Factor 88 de Diferenciación Mieloide/genética , Factor Plaquetario 4/sangre , Factor Plaquetario 4/genética , Trombocitopenia/diagnóstico , Trombocitopenia/virología , Receptores Toll-Like/deficiencia , Receptores Toll-Like/genética , Proteína 3 de Membrana Asociada a Vesículas/sangre , Proteína 3 de Membrana Asociada a Vesículas/genética

RESUMEN

BACKGROUND: We previously reported increased unstimulated blood levels of interferon-gamma in persons with latent tuberculosis infection (LTBI) in the United States, suggesting enhanced immune activation in LTBI. To investigate this further in a TB-endemic setting, we assessed interferon-gamma levels in persons with and without LTBI in Peru. METHODS: We analyzed data from patients with and without a recent type 1 (spontaneous) acute myocardial infarction (AMI) who were enrolled from two public hospital networks in Lima, Peru, and underwent LTBI testing using the QuantiFERON® TB Gold In-tube (QFT) assay. Participants with a positive QFT test were defined as having LTBI, whereas participants with a negative QFT test were defined as non-LTBI. Unstimulated interferon-gamma was quantified via enzyme-linked immunosorbent assay in the QFT nil-tube, which does not contain antigens. We compared unstimulated interferon-gamma levels between LTBI and non-LTBI groups using the Wilcoxon rank sum test. We used proportional odds modeling for multivariable analysis. RESULTS: Data from 214 participants were included in this analysis. Of those, 120 (56%) had LTBI. There were no significant differences in age, sex and comorbidities between LTBI and non-LTBI participants, except for recent AMI that was more frequent in LTBI. LTBI participants had higher unstimulated interferon-gamma levels compared to non-LTBI participants (median, interquartile range; 14 pg/mL, 6.5-52.8 vs. 6.5 pg/mL, 4.5-15; P<0.01). LTBI remained associated with higher unstimulated interferon-gamma levels after controlling for age, sex, recent AMI, history of hypertension, diabetes mellitus, dyslipidemia, end stage renal disease, malignancy, obesity, and tobacco use (adjusted odds ratio, 2.93; 95% confidence interval, 1.8-4.9). In a sensitivity analysis that excluded participants with AMI, the association between unstimulated interferon-gamma and LTBI remained present (adjusted odds ratio; 3.93; 95% confidence interval, 1.9-8.2). CONCLUSIONS: LTBI was associated with higher unstimulated interferon-gamma levels. These data suggest ongoing immune activation in LTBI.

Asunto(s)

Interferón gamma/sangre , Tuberculosis Latente/sangre , Factores de Edad , Anciano , Femenino , Humanos , Tuberculosis Latente/epidemiología , Masculino , Persona de Mediana Edad , Perú/epidemiología , Factores de Riesgo

RESUMEN

Pneumocystis species are fungal pathogens that cause pneumonia in immunocompromised hosts. Lung damage during Pneumocystis pneumonia is predominately due to the inflammatory immune response. Pneumocystis species have a biphasic life cycle. Optimal innate immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, the trophic life cycle stage broadly suppresses proinflammatory responses to multiple pathogen-associated molecular patterns (PAMPs), including ß-1,3-glucan. Little is known about the contribution of these life cycle stages to the development of protective adaptive responses to Pneumocystis infection. Here we report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B-cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised recombination-activating gene 2 knockout (RAG2-/-) mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia.

Asunto(s)

Inmunidad Adaptativa , Inmunidad Innata , Infecciones por Pneumocystis/inmunología , Pneumocystis/inmunología , Animales , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas de Unión al ADN/deficiencia , Huésped Inmunocomprometido , Memoria Inmunológica , Ratones Endogámicos BALB C , Ratones Noqueados

RESUMEN

Background: Tuberculosis has been associated with an increased risk of cardiovascular disease (CVD), including acute myocardial infarction (AMI). We investigated whether latent tuberculosis infection (LTBI) is associated with AMI. Methods: We conducted a case-control study in 2 large national public hospital networks in Lima, Peru, between July 2015 and March 2017. Case patients were patients with a first time diagnosis of type 1 (spontaneous) AMI. Controls were patients without a history of AMI. We excluded patients with known human immunodeficiency virus infection, tuberculosis disease, or prior LTBI treatment. We used the QuantiFERON-TB Gold In-Tube assay to identify LTBI. We used logistic regression modeling to estimate the odds ratio (OR) of LTBI in AMI case patients versus non-AMI controls. Results: We enrolled 105 AMI case patients and 110 non-AMI controls during the study period. Overall, the median age was 62 years (interquartile range, 56-70 years); 69% of patients were male; 64% had hypertension, 40% dyslipidemia, and 39% diabetes mellitus; 30% used tobacco; and 24% were obese. AMI case patients were more likely than controls to be male (80% vs 59%; P < .01) and tobacco users (41% vs 20%; P < .01). LTBI was more frequent in AMI case patients than in controls (64% vs 49% [P = .03]; OR, 1.86; 95% confidence interval [CI], 1.08-3.22). After adjustment for age, sex, hypertension, dyslipidemia, diabetes mellitus, tobacco use, obesity, and family history of coronary artery disease, LTBI remained independently associated with AMI (adjusted OR, 1.90; 95% CI, 1.05-3.45). Conclusions: LTBI was independently associated with AMI. Our results suggest a potentially important role of LTBI in CVD.

Asunto(s)

Tuberculosis Latente/complicaciones , Infarto del Miocardio/complicaciones , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Tuberculosis Latente/diagnóstico , Modelos Logísticos , Masculino , Persona de Mediana Edad , Perú , Factores de Riesgo

RESUMEN

The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including ß-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with ß-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Pneumocystis/crecimiento & desarrollo , Pneumocystis/inmunología , Animales , Presentación de Antígeno , Células Dendríticas/metabolismo , Células Dendríticas/patología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Linfocitos , Ratones , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Neumonía por Pneumocystis/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta-Glucanos/inmunología , beta-Glucanos/metabolismo

RESUMEN

Development of Pneumocystis pneumonia (PCP) is a common problem among immunosuppressed individuals. There are windows of opportunity in which vaccination would be beneficial, but to date, no vaccines have made it to clinical trials. Significant hurdles to vaccine development include host range specificity, making it difficult to translate from animal models to humans. Discovery of cross-reactive epitopes is critical to moving vaccine candidates from preclinical animal studies to clinical trials.

Asunto(s)

Vacunas Fúngicas/inmunología , Pneumocystis carinii/inmunología , Neumonía por Pneumocystis/prevención & control , Animales , Humanos

RESUMEN

Recent studies show a substantial incidence of Pneumocystis jirovecii colonization and infection in patients with chronic inflammatory lung conditions. However, little is known about the impact of Pneumocystis upon the regulation of pulmonary immunity. We demonstrate here that Pneumocystis polarizes macrophages towards an alternatively activated macrophage-like phenotype. Genetically engineered mice that lack the ability to signal through IL-4 and IL-13 were used to show that Pneumocystis alternative macrophage activation is dependent upon signaling through these cytokines. To determine whether Pneumocystis-induced macrophage polarization would impact subsequent immune responses, we infected mice with Pneumocystis and then challenged them with Pseudomonas aeruginosa 14 days later. In co-infected animals, a higher proportion of macrophages in the alveolar and interstitial spaces expressed both classical and alternatively activated markers and produced the regulatory cytokines TGFß and IL-10, as well as higher arginase levels than in mice infected with P. aeruginosa alone. Our results suggest that Pneumocystis reprograms the overall macrophage repertoire in the lung to that of a more alternatively-activated setpoint, thereby altering subsequent immune responses. These data may help to explain the association between Pneumocystis infection and decline in pulmonary function.

Asunto(s)

Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Infecciones por Pneumocystis/inmunología , Infecciones por Pneumocystis/metabolismo , Animales , Biomarcadores , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunofenotipificación , Activación de Macrófagos/inmunología , Ratones , Ratones Noqueados , Fenotipo , Infecciones por Pneumocystis/genética , Infecciones por Pneumocystis/microbiología , Pneumocystis carinii/inmunología , Neumonía Bacteriana/genética , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/microbiología , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética

RESUMEN

The cyst cell wall ß-glucans of Pneumocystis have been shown to stimulate immune responses in lung epithelial cells, dendritic cells, and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with these innate immune cells. Here, we report differences in the responses of both neonatal and adult mice to the trophic and cystic life cycle stages of Pneumocystis murina The adult and neonatal immune responses to infection with Pneumocystis murina trophic forms were less robust than the response to infection with a physiologically normal mixture of cysts and trophic forms. Cysts promoted the recruitment of nonresident innate immune cells and T and B cells into the lungs. Cysts, but not trophic forms, stimulated increased IFN-γ cytokine concentrations in the alveolar spaces, and an increase in IFN-γ-producing CD4+ T cells. In vitro, bone marrow-derived dendritic cells (BMDCs) stimulated with cysts produced the proinflammatory cytokines IL-1ß and IL-6. In contrast, trophic forms suppressed ß-glucan-, LTA-, and LPS-induced IL-1ß, IL-6, and TNFα production by BMDCs and antigen presentation to CD4+ T cells. The negative effects of trophic forms were not due to ligation of mannose receptor. Our results indicate that optimal innate and adaptive immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, trophic forms suppress ß-glucan-induced proinflammatory responses in vitro, suggesting that the trophic forms dampen cyst-induced inflammation in vivo.

RESUMEN

We previously reported that neonatal mice infected with influenza A virus (IAV) develop interstitial pneumonia characterized by reduced lung cytokine and chemokine responses. The failure of T cells to infiltrate the airways of neonates correlated with delayed clearance of sublethal IAV infections compared to adults. We hypothesized that negative regulators in the neonatal lungs such as cytokines or T regulatory (Treg) cells are responsible for these differences. Neonates either deficient in interleukin-10 (IL-10) or with T cells unresponsive to transforming growth factor-ß signaling due to absence of SMAD family member 4 (Smad4) had similar IAV clearance kinetics to wild-type pups and no difference in T-cell responses. In contrast, functional depletion of Treg cells with anti-CD25 monoclonal antibody resulted in increased proportions of activated CD4(+) T cells in the lungs, but failure to clear IAV. Similarly, scurfy pups (mutation in forkhead box P3 [Foxp3] rendering them deficient in Treg cells) had increased proportions of activated T cells in the lungs compared to littermate controls. Scurfy pups also had increased proportions of IL-13-producing CD4(+) T cells. Interestingly, like anti-CD25-treated pups, scurfy pups had significantly elevated viral loads compared to controls. Based on these data, we conclude that Tregs are critical for clearance of IAV in neonatal mice.

Asunto(s)

Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T Reguladores/inmunología , Animales , Animales Recién Nacidos , Femenino , Humanos , Pulmón/patología , Pulmón/virología , Masculino , Ratones Endogámicos C57BL , Carga Viral

RESUMEN

B cells play a critical role in the clearance of Pneumocystis. In addition to production of Pneumocystis-specific Abs, B cells are required during the priming phase for CD4(+) T cells to expand normally and generate memory. Clearance of Pneumocystis was found to be dependent on Ag specific B cells and on the ability of B cells to secrete Pneumocystis-specific Ab, as mice with B cells defective in these functions or with a restricted BCR were unable to control Pneumocystis infection. Because Pneumocystis-specific antiserum was only able to partially protect B cell-deficient mice from infection, we hypothesized that optimal T cell priming requires fully functional B cells. Using adoptive transfer and B cell depletion strategies, we determined that optimal priming of CD4(+) T cells requires B cells during the first 2-3 d of infection and that this was independent of the production of Ab. T cells that were removed from Pneumocystis-infected mice during the priming phase were fully functional and able to clear Pneumocystis infection upon adoptive transfer into Rag1(-/-) hosts, but this effect was ablated in mice that lacked fully functional B cells. Our results indicate that T cell priming requires a complete environment of Ag presentation and activation signals to become fully functional in this model of Pneumocystis infection.

Asunto(s)

Anticuerpos Antifúngicos/biosíntesis , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Pneumocystis/inmunología , Neumonía por Pneumocystis/inmunología , Traslado Adoptivo , Animales , Linfocitos B/microbiología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/microbiología , Proliferación Celular , Eliminación de Gen , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Inmunidad Humoral , Activación de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía por Pneumocystis/microbiología , Neumonía por Pneumocystis/patología , Neumonía por Pneumocystis/terapia

RESUMEN

Tissue-resident memory CD8 T cells are a unique subset of virus-specific CTLs that bolster local immune responses after becoming lodged in previously infected tissues. These cells provide enhanced protection by intercepting returning pathogens before a new infection gets established. In contrast, central memory CD8 T cells circulate in the bloodstream and proliferate in secondary lymphoid organs before replenishing effector and memory CD8 T cell populations in remote parts of the body. Both populations of virus-specific memory CD8 T cells participate in immunity to influenza virus infection; however, the signaling pathways that instruct developing memory CD8 T cells to distribute to specific tissues are poorly defined. We show that TGF-ß promotes the development of pulmonary tissue-resident memory T cells via a signaling pathway that does not require the downstream signaling intermediate Sma- and Mad-related protein (Smad)4. In contrast, circulating memory CD8 T cells have no requirement for TGF-ß but show signs of arrested development in the absence of Smad4, including aberrant CD103 expression. These signaling pathways alter the distribution of virus-specific CTLs in the lungs but do not prevent robust cytokine responses. Our data show that Smad4 is required for normal differentiation of multiple subsets of virus-specific CD8 T cells. In normal circumstances, Smad4 may be activated via a pathway that bypasses the TGF-ß receptor. Improved understanding of these signaling pathways could be used to augment vaccine-induced immunity.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Linaje de la Célula/inmunología , Memoria Inmunológica , Pulmón/inmunología , Proteína Smad4/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Diferenciación Celular , Regulación de la Expresión Génica , Virus de la Influenza A/inmunología , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/inmunología , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/virología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Transducción de Señal , Proteína Smad4/deficiencia , Proteína Smad4/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/farmacología , Quimera por Trasplante

RESUMEN

The burden of tuberculosis and cardiovascular disease (CVD) is enormous worldwide. CVD rates are rapidly increasing in low- and middle-income countries. Public health programs have been challenged with the overlapping tuberculosis and CVD epidemics. Monocyte/macrophages, lymphocytes and cytokines involved in cellular mediated immune responses against Mycobacterium tuberculosis are also main drivers of atherogenesis, suggesting a potential pathogenic role of tuberculosis in CVD via mechanisms that have been described for other pathogens that establish chronic infection and latency. Studies have shown a pro-atherogenic effect of antibody-mediated responses against mycobacterial heat shock protein-65 through cross reaction with self-antigens in human vessels. Furthermore, subsets of mycobacteria actively replicate during latent tuberculosis infection (LTBI), and recent studies suggest that LTBI is associated with persistent chronic inflammation that may lead to CVD. Recent epidemiologic work has shown that the risk of CVD in persons who develop tuberculosis is higher than in persons without a history of tuberculosis, even several years after recovery from tuberculosis. Together, these data suggest that tuberculosis may play a role in the pathogenesis of CVD. Further research to investigate a potential link between tuberculosis and CVD is warranted.

RESUMEN

Pneumocystis species are opportunistic fungal pathogens that induce tumor necrosis factor (TNF) production by alveolar macrophages. Here we report that B cells from the draining lymph nodes as well as lung CD4(+) T cells are important producers of TNF upon Pneumocystis murina infection. To determine the importance of B cell-derived TNF in the primary response to P. murina, we generated bone marrow chimeras whose B cells were unable to produce TNF. The lung P. murina burden at 10 days postinfection in TNF knockout (TNFKO) chimeras was significantly higher than that in wild-type (WT) chimeras, which corresponded to reduced numbers of activated CD4(+) T cells in the lungs at this early time point. Furthermore, CD4(+) T cells isolated from P. murina-infected TNFKO chimeras were unable to stimulate clearance of P. murina upon adoptive transfer to recombinase-deficient (RAG1KO) hosts. Together, these data indicate that B cell-derived TNF plays an important function in promoting CD4(+) T cell expansion and production of TNF and facilitating protection against P. murina infection.

Asunto(s)

Linfocitos B/inmunología , Pneumocystis/inmunología , Neumonía por Pneumocystis/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/genética