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
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
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 ViralRESUMEN
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/terapiaRESUMEN
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/genéticaRESUMEN
Pneumocystis pneumonia was first diagnosed in malnourished children and has more recently been found in children with upper respiratory symptoms. We previously reported that there is a significant delay in the immune response in newborn mice infected with Pneumocystis compared to adults (Garvy BA, Harmsen AG, Infect. Immun. 64:3987-3992, 1996, and Garvy BA, Qureshi M, J. Immunol. 165:6480-6486, 2000). This delay is characterized by the failure of neonatal lungs to upregulate proinflammatory cytokines and attract T cells into the alveoli. Here, we report that regardless of the age at which we infected the mice, they failed to mount an inflammatory response in the alveolar spaces until they were 21 days of age or older. Anti-inflammatory cytokines had some role in dampening inflammation, since interleukin-10 (IL-10)-deficient pups cleared Pneumocystis faster than wild-type pups and the neutralization of transforming growth factor beta (TGF-ß) with specific antibody enhanced T cell migration into the lungs at later time points. However, the clearance kinetics were similar to those of control pups, suggesting that there is an intrinsic deficiency in the ability of innate immunity to control Pneumocystis. We found, using an adoptive transfer strategy, that the lung environment contributes to association of Pneumocystis organisms with alveolar macrophages, implying no intrinsic deficiency in the binding of Pneumocystis by neonatal macrophages. Using both in vivo and in vitro assays, we found that Pneumocystis organisms were less able to stimulate translocation of NF-κB to the nucleus of alveolar macrophages from neonatal mice. These data indicate that there is an early unresponsiveness of neonatal alveolar macrophages to Pneumocystis infection that is both intrinsic and related to the immunosuppressive environment found in neonatal lungs.
Asunto(s)
Inmunidad Innata/fisiología , Macrófagos Alveolares/microbiología , Infecciones por Pneumocystis/microbiología , Pneumocystis/clasificación , Animales , Animales Recién Nacidos , Líquido del Lavado Bronquioalveolar/química , Regulación de la Expresión Génica/inmunología , Inmunoglobulina G/análisis , Inmunoglobulina G/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Ganglios Linfáticos , Ratones , Ratones Endogámicos , Infecciones por Pneumocystis/inmunología , Alveolos Pulmonares/microbiología , Linfocitos T/fisiología , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Influenza virus is a significant cause of mortality and morbidity in children; however, little is known about the T cell response in infant lungs. Neonatal mice are highly vulnerable to influenza and only control very low doses of virus. We compared the T cell response to influenza virus infection between mice infected as adults or at 2 d old and observed defective migration into the lungs of the neonatal mice. In the adult mice, the numbers of T cells in the lung interstitia peaked at 10 d postinfection, whereas neonatal T cell infiltration, activation, and expression of TNF-alpha was delayed until 2 wk postinfection. Although T cell numbers ultimately reached adult levels in the interstitia, they were not detected in the alveoli of neonatal lungs. Instead, the alveoli contained eosinophils and neutrophils. This altered infiltrate was consistent with reduced or delayed expression of type 1 cytokines in the neonatal lung and differential chemokine expression. In influenza-infected neonates, CXCL2, CCL5, and CCL3 were expressed at adult levels, whereas the chemokines CXCL1, CXCL9, and CCL2 remained at baseline levels, and CCL11 was highly elevated. Intranasal administration of CCL2, IFN-gamma, or CXCL9 was unable to draw the neonatal T cells into the airways. Together, these data suggest that the T cell response to influenza virus is qualitatively different in neonatal mice and may contribute to an increased morbidity.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Virus de la Influenza A/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virología , Animales , Animales Recién Nacidos , Proliferación Celular , Eosinófilos/inmunología , Eosinófilos/patología , Eosinófilos/virología , Femenino , Predisposición Genética a la Enfermedad , Macrófagos/inmunología , Macrófagos/patología , Macrófagos/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/patología , Neutrófilos/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , Subgrupos de Linfocitos T/patologíaRESUMEN
Alveolar macrophages are the effector cells largely responsible for clearance of Pneumocystis carinii from the lungs. Binding of organisms to beta-glucan and mannose receptors has been shown to stimulate phagocytosis of the organisms. To further define the mechanisms used by alveolar macrophages for clearance of P. carinii, mice deficient in the expression of scavenger receptor A (SRA) were infected with P. carinii, and clearance of organisms was monitored over time. SRA-deficient (SRAKO) mice consistently cleared P. carinii faster than did wild-type control mice. Expedited clearance corresponded to elevated numbers of activated CD4(+) T cells in the alveolar spaces of SRAKO mice compared to wild-type mice. Alveolar macrophages from SRAKO mice had increased expression of CD11b on their surfaces, consistent with an activated phenotype. However, they were not more phagocytic than macrophages expressing SRA, as measured by an in vivo phagocytosis assay. SRAKO alveolar macrophages produced significantly more tumor necrosis factor alpha (TNF-alpha) than wild-type macrophages when stimulated with lipopolysaccharide in vitro but less TNF-alpha in response to P. carinii in vitro. However, upon in vivo stimulation, SRAKO mice produced significantly more TNF-alpha, interleukin 12 (IL-12), and IL-18 in response to P. carinii infection than did wild-type mice. Together, these data indicate that SRA controls inflammatory cytokines produced by alveolar macrophages in the context of P. carinii infection.
Asunto(s)
Pneumocystis carinii/inmunología , Neumonía por Pneumocystis/inmunología , Receptores Depuradores de Clase A/inmunología , Animales , Antígeno CD11b/análisis , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Inflamación/inmunología , Interleucina-12/biosíntesis , Interleucina-18/biosíntesis , Pulmón/inmunología , Macrófagos Alveolares/química , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Fagocitosis , Receptores Depuradores de Clase A/genética , Subgrupos de Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis pulmonary infections. We have previously demonstrated that there is approximately a 3-week delay in the clearance of Pneumocystis organisms from pup mouse lungs compared to that in adults. We have further shown that there is approximately a 1-week delay in alveolar macrophage activation in pups versus adult mice. Alveolar macrophages are the primary effector cells responsible for the killing and clearance of Pneumocystis, suggesting that pup alveolar macrophages may be involved in the delayed clearance of this organism. Alveolar macrophages cultured in vitro with Pneumocystis alone demonstrate little to no activation, as indicated by a lack of cytokine production. However, when cultured with lipopolysaccharide (LPS) or zymosan, cytokine production was markedly increased, suggesting that pup alveolar macrophages are specifically unresponsive to Pneumocystis organisms rather than being intrinsically unable to become activated. Furthermore, pup mice treated with aerosolized, heat-killed Escherichia coli in vivo were able to clear Pneumocystis more efficiently than were control mice. Together, these data suggest that while pup alveolar macrophages are unresponsive to P. carinii f. sp. muris organisms, they are capable of activation by heat-killed E. coli in vivo, as well as LPS and zymosan in vitro. The lack of response of pup mice to P. carinii f. sp. muris may reflect protective mechanisms specific to the developing pup lung, but ultimately it results in insufficient clearance of Pneumocystis organisms.
Asunto(s)
Escherichia coli/inmunología , Calor , Pulmón/microbiología , Activación de Macrófagos/inmunología , Pneumocystis carinii/aislamiento & purificación , Neumonía por Pneumocystis/inmunología , Animales , Animales Recién Nacidos , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/metabolismo , Escherichia coli/crecimiento & desarrollo , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Fagocitosis , Neumonía por Pneumocystis/microbiologíaRESUMEN
B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica , Activación de Linfocitos/inmunología , Pneumocystis carinii/inmunología , Neumonía por Pneumocystis/inmunología , Animales , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/trasplante , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/genética , Cambio de Clase de Inmunoglobulina , Isotipos de Inmunoglobulinas/biosíntesis , Cooperación Linfocítica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Neumonía por Pneumocystis/microbiologíaRESUMEN
Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to infection with Pneumocystis. We have previously shown that there is a significant delay in clearance of the organisms from the lungs of neonatal mice compared to adults. Since alveolar macrophages are the effector cells responsible for killing and clearance of Pneumocystis, we have examined alveolar macrophage activity in neonatal mice. We found that alveolar macrophage activation is delayed about 1 week in Pneumocystis-infected neonates compared to adults. Opsonization of the organism by Pneumocystis-specific antibody resulted in increased clearance of the organism in neonatal mice; however, there was decreased expression of activation markers on neonatal alveolar macrophages and reduced levels of cytokines associated with macrophage activation. Mice born to immunized dams had significant amounts of Pneumocystis-specific immunoglobulin G in their lungs and serum at day 7 postinfection, whereas mice born to naive dams had merely detectable levels. This difference correlated with enhanced Pneumocystis clearance in mice born to immunized dams. The increase in specific antibody, however, did not result in significant inflammation in the lungs, as no differences in numbers of activated CD4+ cells were observed. Furthermore, there was no difference in cytokine or chemokine concentrations in the lungs of pups born to immune compared to naive dams. These findings indicate that specific antibody plays an important role in Pneumocystis clearance from lungs of infected neonates; moreover, this process occurs without inducing inflammation in the lungs.
Asunto(s)
Animales Recién Nacidos/inmunología , Anticuerpos Antibacterianos/administración & dosificación , Inmunización Pasiva , Inflamación/inmunología , Pneumocystis carinii/inmunología , Neumonía por Pneumocystis/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inflamación/fisiopatología , Pulmón/inmunología , Activación de Macrófagos , Macrófagos Alveolares/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Neumonía por Pneumocystis/inmunología , Neumonía por Pneumocystis/microbiología , Neumonía por Pneumocystis/fisiopatologíaRESUMEN
Both CD4(+) T cells and B cells are critical for defense against Pneumocystis carinii infection; however, the mechanism by which B cells mediate protection is unknown. We show that P. carinii-specific IgM is not sufficient to mediate clearance of P. carinii from the lungs since CD40-deficient mice produced normal levels of specific IgM, but were unable to clear the organisms. Using chimeric mice in which the B cells were deficient in CD40 (CD40KO chimeras) we found that clearance of P. carinii infection is delayed compared with wild-type controls. These CD40KO chimeric mice produced normal levels of P. carinii-specific IgM, but did not produce class-switched IgG or IgA. Similarly, clearance of P. carinii was delayed in mice deficient in FcgammaRI and III (FcgammaRKO), indicating that P. carinii-specific IgG partially mediates opsonization and clearance of P. carinii. Opsonization of organisms by complement did not compensate for the lack of specific IgG or FcgammaR, since C3-deficient and C3-depleted FcgammaRKO mice were still able to clear P. carinii. Finally, micro MT and CD40KO chimeric mice had reduced numbers of activated CD4(+) T cells in the lungs and lymph nodes compared with wild-type mice, suggesting that B cells are important for activation of T cells in response to P. carinii. Together these data indicate that P. carinii-specific IgG plays an important, but not critical, role in defense against P. carinii. Moreover, these data suggest that B cells also mediate host defense against P. carinii by facilitating CD4(+) T cell activation or expansion.