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INTRODUCTION: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. METHODS: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. RESULTS: Within iNKT-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT-/- mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. CONCLUSIONS: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.
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Macrófagos Peritoneais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais , Sepse , Animais , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Sepse/imunologia , Sepse/microbiologia , Células T Matadoras Naturais/imunologia , Camundongos , Masculino , Superóxidos/metabolismo , Citocinas/metabolismo , Modelos Animais de DoençasRESUMO
Introduction: Sepsis remains a major source of morbidity and mortality in neonates, and characterization of immune regulation in the neonatal septic response remains limited. HVEM is a checkpoint regulator which can both stimulate or inhibit immune responses and demonstrates altered expression after sepsis. We hypothesized that signaling via HVEM would be essential for the neonatal response to sepsis, and that therefore blockade of this pathway would improve survival to septic challenge. Methods: To explore this, neonatal mice were treated with cecal slurry (CS), CS with Anti-HVEM antibody (CS-Ab) or CS with isotype (CS-IT) and followed for 7-day survival. Mice from all treatment groups had thymus, lung, kidney and peritoneal fluid harvested, weighed, and stained for histologic evaluation, and changes in cardiac function were assessed with echocardiography. Results: Mortality was significantly higher for CS-Ab mice (72.2%) than for CS-IT mice (22.2%). CS resulted in dysregulated alveolar remodeling, but CS-Ab lungs demonstrated significantly less dysfunctional alveolar remodeling than CS alone (MCL 121.0 CS vs. 87.6 CS-Ab), as well as increased renal tubular vacuolization. No morphologic differences in alveolar septation or thymic karyorrhexis were found between CS-Ab and CS-IT. CS-Ab pups exhibited a marked decrease in heart rate (390.3 Sh vs. 342.1 CS-Ab), stroke volume (13.08 CS-IT vs. 8.83 CS-Ab) and ultimately cardiac output (4.90 Sh vs. 3.02 CS-Ab) as well as a significant increase in ejection fraction (73.74 Sh vs. 83.75 CS-Ab) and cardiac strain (40.74 Sh vs. 51.16 CS-Ab) as compared to CS-IT or Sham animals. Discussion: While receptor ligation of aspects of HVEM signaling, via antibody blockade, appears to mitigate aspects of lung injury and thymic involution, stimulatory signaling via HVEM still seems to be necessary for vascular and hemodynamic resilience and overall neonatal mouse survival in response to this experimental polymicrobial septic insult. This dissonance in the activity of anti-HVEM neutralizing antibody in neonatal animals speaks to the differences in how septic cardiac dysfunction should be considered and approached in the neonatal population.
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Animais Recém-Nascidos , Sepse Neonatal , Transdução de Sinais , Animais , Camundongos , Sepse Neonatal/imunologia , Sepse Neonatal/mortalidade , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/imunologia , Modelos Animais de Doenças , Feminino , Cardiopatias/etiologia , Cardiopatias/imunologia , Pulmão/imunologia , Pulmão/patologia , Sepse/imunologia , Sepse/metabolismoRESUMO
BACKGROUND: iNKT-cells are innate regulatory lymphocytes capable of directing immune and inflammatory responses to sepsis. Repeat stimulation of iNKT-cells leads to the induction of anergy with the emergence of a hyporesponsive CD3low iNKT-cell subpopulation. METHODS: iNKT-cells were isolated from critical ill surgical patients with sepsis and phenotyped for CD3 expression. This was correlated with degree of severity of illness, as denoted by APACHE-II score. RESULTS: Comparing healthy volunteers to critically ill septic patients, it was noted that increasing severity of sepsis was associated with increasing frequency of circulating CD3low-iNKT-cell populations. CONCLUSION: The emergence of CD3low -iNKT-cells may serve as a clinically translatable marker of degree of sepsis-induced immune dysfunction.
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Estado Terminal , Sepse , Humanos , LinfócitosRESUMO
Sepsis is a dysregulated systemic immune response to infection that is responsible for â¼35% of in-hospital deaths at a significant fiscal health care cost. Our laboratory, among others, has demonstrated the efficacy of targeting negative checkpoint regulators (NCRs) to improve survival in a murine model of sepsis, cecal ligation and puncture (CLP). B7-CD28 superfamily member, V-domain Immunoglobulin Suppressor of T cell Activation (VISTA), is an ideal candidate for strategic targeting in sepsis. VISTA is a 35-45â kDa type 1 transmembrane protein with unique biology that sets it apart from all other NCRs. We recently reported that VISTA-/- mice had a significant survival deficit post CLP which was rescued upon adoptive transfer of a VISTA-expressing pMSCV-mouse Foxp3-EF1α-GFP-T2A-puro stable Jurkat cell line (Jurkatfoxp3 T cells). Based on our prior study, we investigated the effector cell target of Jurkatfoxp3 T cells in VISTA-/- mice. γδ T cells are a powerful lymphoid subpopulation that require regulatory fine-tuning by Tregs to prevent overt inflammation/pathology. In this study, we hypothesized that Jurkatfoxp3 T cells non-redundantly modulate the γδ T cell population post CLP. We found that VISTA-/- mice have an increased accumulation of intestinal CD69low γδ T cells which are not protective in murine sepsis. Adoptive transfer of Jurkatfoxp3 T cells, decreased the intestinal γδ T cell population, suppressed proliferation, skewed remaining γδ T cells toward a CD69high phenotype, and increased sCD40L in VISTA-/- mice post CLP. These results support a potential regulatory mechanism by which VISTA skews intestinal γδ T cell lineage representation in murine sepsis.
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ABSTRACT: Background: Sepsis is marked by a dysregulated immune response to an infection. Invariant natural killer T cells ( i NKT cells) are a pluripotent lymphocyte subpopulation capable of affecting and coordinating the immune response to sepsis. The spleen is an important site of immune interactions in response to an infection. Splenic i NKT cells have emerged as important potential frontline mediators of chronic immune response. There are few data addressing the role splenic of i NKT cells in response to intra-abdominal polymicrobial sepsis. Methods: The cecal ligation and puncture model was used to create intra-abdominal sepsis in 8- to 12-week-old wild-type, i NKT -/- , or programmed cell death receptor-1 (PD-1) -/- mice. Twenty-four hours later, spleens were harvested. Flow cytometry was used for phenotyping using monoclonal antibodies. Cell sort was used to isolate i NKT cells. A macrophage cell line was used to assess i NKT cell-phagocyte interactions. Enzyme-linked immunosorbent assay was used for cytokine analysis. Results: Splenic i NKT-cell populations rapidly declined following induction of sepsis. Within i NKT-cell -/- mice, a distinct baseline hyperinflammatory environment was noted. Within wild type, sepsis induced an increase in splenic IL-6 and IL-10, whereas in i NKT -/- mice, there was no change in elevated IL-6 levels and a noted decrease in IL-10 expression. Further, following sepsis, PD-1 expression was increased upon spleen i NKT cells. With respect to PD-1 ligands upon phagocytes, PD-1 ligand expression was unaffected, whereas PD-L2 expression was significantly affected by the presence of PD-1. Conclusions: Invariant natural killer T cells play a distinct role in the spleen response to sepsis, an effect mediated by the checkpoint protein PD-1. Given that modulators are available in clinical trials, this offers a potential therapeutic target in the setting of sepsis-induced immune dysfunction.
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Células T Matadoras Naturais , Sepse , Animais , Camundongos , Receptor de Morte Celular Programada 1 , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Baço , Sepse/metabolismoRESUMO
Introduction: The co-regulatory molecule, HVEM, can stimulate or inhibit immune function, but when co-expressed with BTLA, forms an inert complex preventing signaling. Altered HVEM or BTLA expression, separately have been associated with increased nosocomial infections in critical illness. Given that severe injury induces immunosuppression, we hypothesized that varying severity of shock and sepsis in murine models and critically ill patients would induce variable increases in HVEM/BTLA leukocyte co-expression. Methods: In this study, varying severities of murine models of critical illness were utilized to explore HVEM+BTLA+ co-expression in the thymic and splenic immune compartments, while circulating blood lymphocytes from critically ill patients were also assessed for HVEM+BTLA+ co-expression. Results: Higher severity murine models resulted in minimal change in HVEM+BTLA+ co-expression, while the lower severity model demonstrated increased HVEM+BTLA+ co-expression on thymic and splenic CD4+ lymphocytes and splenic B220+ lymphocytes at the 48-hour time point. Patients demonstrated increased co-expression of HVEM+BTLA+ on CD3+ lymphocytes compared to controls, as well as CD3+Ki67- lymphocytes. Both L-CLP 48hr mice and critically ill patients demonstrated significant increases in TNF-α. Discussion: While HVEM increased on leukocytes after critical illness in mice and patients, changes in co-expression did not relate to degree of injury severity of murine model. Rather, co-expression increases were seen at later time points in lower severity models, suggesting this mechanism evolves temporally. Increased co-expression on CD3+ lymphocytes in patients on non-proliferating cells, and associated TNF-α level increases, suggest post-critical illness co-expression does associate with developing immune suppression.
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Introduction: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a commonly occurring sequelae of traumatic injury resulting from indirect insults like hypovolemic shock and/or extrapulmonary sepsis. The high lethality rate associated with these pathologies outlines the importance of clarifying the "priming" effects seen in the post-shock lung microenvironment, which are understood to bring about a dysregulated or overt immune response when triggered by a secondary systemic infectious/septic challenge culminating in ALI. In this pilot project, we test the hypothesis that application of a single cell multiomics approach can elucidate novel phenotype specific pathways potentially contributing to shock-induced ALI/ARDS. Methods: Hypovolemic shock was induced in C57BL/6 (wild-type), PD-1, PD-L1, or VISTA gene deficient male mice, 8-12 weeks old. Wild-type sham surgeries function as negative controls. A total of 24-h post-shock rodents were sacrificed, their lungs harvested and sectioned, with pools prepared from 2 mice per background, and flash frozen on liquid nitrogen. N = 2 biological replicates (representing 4 mice total) were achieved for all treatment groups across genetic backgrounds. Samples were received by the Boas Center for Genomics and Human Genetics, where single cell multiomics libraries were prepared for RNA/ATAC sequencing. The analysis pipeline Cell Ranger ARC was implemented to attain feature linkage assessments across genes of interest. Results: Sham (pre-shock) results suggest high chromatin accessibility around calcitonin receptor like receptor (CALCRL) across cellular phenotypes with 17 and 18 feature links, exhibiting positive correlation with gene expression between biological replicates. Similarity between both sample chromatin profiles/linkage arcs is evident. Post-shock wild-type accessibility is starkly reduced across replicates where the number of feature links drops to 1 and 3, again presenting similar replicate profiles. Samples from shocked gene deficient backgrounds displayed high accessibility and similar profiles to the pre-shock lung microenvironment. Conclusion: High pre-shock availability of DNA segments and their positive correlation with CALCRL gene expression suggests an apparent regulatory capacity on transcription. Post-shock gene deficient chromatin profiles presented similar results to that of pre-shock wild-type samples, suggesting an influence on CALCRL accessibility. Key changes illustrated in the pre-ALI context of shock may allow for additional resolution of "priming" and "cellular pre-activation/pre-disposition" processes within the lung microenvironment.
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Sepsis, a dysfunctional immune response to infection leading to life-threatening organ injury, represents a significant global health issue. Neonatal sepsis is disproportionately prevalent and has a cost burden of 2-3 times that of adult patients. Despite this, no widely accepted definition for neonatal sepsis or recommendations for management exist and those created for pediatric patients are significantly limited in their applicability to this unique population. This is in part due to neonates' reliance on an innate immune response (which is developmentally more prominent in the neonate than the immature adaptive immune response) carried out by dysfunctional immune cells, including neutrophils, antigen-presenting cells such as macrophages/monocytes, dendritic cells, etc., natural killer cells, and innate lymphoid regulatory cell sub-sets like iNKT cells, γδ T-cells, etc. Immune checkpoint inhibitors are a family of proteins with primarily suppressive/inhibitory effects on immune and tumor cells and allow for the maintenance of self-tolerance. During sepsis, these proteins are often upregulated and are thought to contribute to the long-term immunosuppression seen in adult patients. Several drugs targeting checkpoint inhibitors, including PD-1 and PD-L1, have been developed and approved for the treatment of various cancers, but no such therapeutics have been approved for the management of sepsis. In this review, we will comparatively discuss the role of several checkpoint inhibitor proteins, including PD-1, PD-L1, VISTA, and HVEM, in the immune response to sepsis in both adults and neonates, as well as posit how they may uniquely propagate their actions through the neonatal innate immune response. We will also consider the possibility of leveraging these proteins in the clinical setting as potential therapeutics/diagnostics that might aid in mitigating neonatal septic morbidity/mortality.
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Sepse Neonatal , Sepse , Adulto , Antígeno B7-H1 , Criança , Humanos , Imunidade Inata , Recém-Nascido , Células Matadoras Naturais , Receptor de Morte Celular Programada 1/fisiologia , Sepse/diagnósticoRESUMO
Sepsis is a systemic immune response to infection that is responsible for ~35% of in-hospital deaths and over 24 billion dollars in annual treatment costs. Strategic targeting of non-redundant negative immune checkpoint protein pathways can cater therapeutics to the individual septic patient and improve prognosis. B7-CD28 superfamily member V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an ideal candidate for strategic targeting in sepsis. We hypothesized that immune checkpoint regulator, VISTA, controls T-regulatory cells (Treg), in response to septic challenge, thus playing a protective role/reducing septic morbidity/mortality. Further, we investigated if changes in morbidity/mortality are due to a Treg-mediated effect during the acute response to septic challenge. To test this, we used the cecal ligation and puncture model as a proxy for polymicrobial sepsis and assessed the phenotype of CD4+ Tregs in VISTA-gene deficient (VISTA-/-) and wild-type mice. We also measured changes in survival, soluble indices of tissue injury, and circulating cytokines in the VISTA-/- and wild-type mice. We found that in wild-type mice, CD4+ Tregs exhibit a significant upregulation of VISTA which correlates with higher Treg abundance in the spleen and small intestine following septic insult. However, VISTA-/- mice have reduced Treg abundance in these compartments met with a higher expression of Foxp3, CTLA4, and CD25 compared to wild-type mice. VISTA-/- mice also have a significant survival deficit, higher levels of soluble indicators of liver injury (i.e., ALT, AST, bilirubin), and increased circulating proinflammatory cytokines (i.e., IL-6, IL-10, TNFα, IL-17F, IL-23, and MCP-1) following septic challenge. To elucidate the role of Tregs in VISTA-/- sepsis mortality, we adoptively transferred VISTA-expressing Tregs into VISTA-/- mice. This adoptive transfer rescued VISTA-/- survival to wild-type levels. Taken together, we propose a protective Treg-mediated role for VISTA by which inflammation-induced tissue injury is suppressed and improves survival in early-stage murine sepsis. Thus, enhancing VISTA expression or adoptively transferring VISTA+ Tregs in early-stage sepsis may provide a novel therapeutic approach to ameliorate inflammation-induced death.
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Proteínas de Checkpoint Imunológico , Sepse , Animais , Citocinas/metabolismo , Humanos , Inflamação , Camundongos , Linfócitos T ReguladoresRESUMO
INTRODUCTION: Severe hemorrhage (Hem) has been shown to be causal for the development of extra-pulmonary/indirect acute respiratory distress syndrome (iARDS) and is associated with severe endothelial cell (EC) injury. EC growth factors, Angiopoietin (Ang)-1 and -2, maintain vascular homeostasis via tightly regulated competitive interaction with the tyrosine kinase receptor, Tie2, expressed on ECs. OBJECTIVE: Since it has been reported that the orphan receptor, Tie1, may be able to play a role in Ang:Tie2 signaling; we chose to examine Tie1's capacity to alter the lung Ang:Tie2 interaction in response to the sequential insults of shock/sepsis (cecal ligation and puncture [CLP]), culminating in iARDS. METHODS: Male mice were subjected to Hem alone or sequential Hem followed 24âhours later by CLP that induces iARDS. Changes in lung and/or plasma levels of Tie1, Tie2, Ang-1, Ang-2, various systemic cytokine/chemokines and indices of lung injury/inflammation were then determined. The role of Tie1 was established by intravenous administration of Tie1 specific or control siRNA at 1 h post-Hem. Alternatively, the contribution of neutrophils was assessed by pre-treating mice with anti-neutrophil antibody depletion 48âh prior to Hem. RESULTS: Lung tissue levels of Tie1 expression elevated over the first 6 to 24âh post-Hem alone. Subsequently, we found that treatment of Hem/CLP mice with Tie1-specific siRNA not only decreased Tie1 expression in lung tissue compared to control siRNA, but, suppressed the rise in lung inflammatory cytokines, lung MPO and the rise in lung protein leak. Finally, much as we have previously shown that neutrophil interaction with resident pulmonary vascular ECs contribute significantly to Ang-2 release and EC dysfunction, central to the development of iARDS. Here, we report that depletion of neutrophils also decreased lung tissue Tie1 expression and increased Tie2 activation in Hem/CLP mice. CONCLUSION: Together, these data imply that shock-induced increased expression of Tie1 can contribute to EC activation by inhibiting Ang:Tie2 interaction, culminating in EC dysfunction and the development of iARDS.
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Pneumonia , Receptor de TIE-1/metabolismo , Síndrome do Desconforto Respiratório , Sepse , Animais , Citocinas/metabolismo , Hemorragia , Inflamação/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Neutrófilos/metabolismo , Pneumonia/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Mammalian cells do not produce chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), although chitin is a structural component of the cell wall of pathogenic microorganisms such as Candida albicans. Mammalian cells, including cells of the innate immune system elaborate chitinases, including chitotriosidase (Chit1), which may play a role in the anti-fungal immune response. In the current study, using knockout mice, we determined the role of Chit1 against systemic candidiasis. Chit1-deficient mice showed significant decrease in kidney fungal burden compared to mice expressing the functional enzyme. Using in vitro anti-candidal neutrophil functional assays, the introduction of the Chit1:chitin digestion end-product, chitobiose (N-acetyl-D-glucosamine dimer, GlcNAc2), decreased fungal-induced neutrophil swarming and Candida killing in vitro. Also, a role for the lectin-like binding site on the neutrophil integrin CR3 (Mac-1, CD11b/CD18) was found through physiological competitive interference by chitobiose. Furthermore, chitobiose treatment of wild type mice during systemic candidiasis resulted in the significant increase in fungal burden in the kidney. These data suggest a counterproductive role of Chit1 in mounting an efficient anti-fungal defense against systemic candidiasis.
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Candidíase/imunologia , Hexosaminidases/fisiologia , Animais , Candidíase/enzimologia , Dissacarídeos/farmacologia , Modelos Animais de Doenças , Feminino , Antígeno de Macrófago 1/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/imunologia , Índice de Gravidade de DoençaRESUMO
Morbidity and mortality associated with neonatal sepsis remains a healthcare crisis. PD1-/- neonatal mice endured experimental sepsis, in the form of cecal slurry (CS), and showed improved rates of survival compared to wildtype (WT) counterparts. End-organ injury, particularly of the lung, contributes to the devastation set forth by neonatal sepsis. PDL1-/- neonatal mice, in contrast to PD1-/- neonatal mice did not have a significant improvement in survival after CS. Because of this, we focused subsequent studies on the impact of PD1 gene deficiency on lung injury. Here, we observed that at 24 h post-CS (but not at 4 or 12 h) there was a marked increase in pulmonary edema (PE), neutrophil influx, myeloperoxidase (MPO) levels, and cytokine expression sham (Sh) WT mice. Regarding pulmonary endothelial cell (EC) adhesion molecule expression, we observed that Zona occludens-1 (ZO-1) within the cell shifted from a membranous location to a peri-nuclear location after CS in WT murine cultured ECs at 24hrs, but remained membranous among PD1-/- lungs. To expand the scope of this inquiry, we investigated human neonatal lung tissue. We observed that the lungs of human newborns exposed to intrauterine infection had significantly higher numbers of PD1+ cells compared to specimens who died from non-infectious causes. Together, these data suggest that PD1/PDL1, a pathway typically thought to govern adaptive immune processes in adult animals, can modulate the largely innate neonatal pulmonary immune response to experimental septic insult. The potential future significance of this area of study includes that PD1/PDL1 checkpoint proteins may be viable therapeutic targets in the septic neonate.
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Antígeno B7-H1/metabolismo , Lesão Pulmonar/etiologia , Pulmão/metabolismo , Sepse Neonatal/complicações , Receptor de Morte Celular Programada 1/metabolismo , Animais , Animais Recém-Nascidos , Antígeno B7-H1/genética , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Humanos , Imunidade Inata , Recém-Nascido , Pulmão/imunologia , Pulmão/patologia , Lesão Pulmonar/imunologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sepse Neonatal/imunologia , Sepse Neonatal/metabolismo , Sepse Neonatal/microbiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor de Morte Celular Programada 1/genética , Edema Pulmonar/etiologia , Edema Pulmonar/imunologia , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Background: Sepsis-related mortality is driven by immune dysfunction. A bidirectional micro-organism-immune cell cross talks exists. Gut Bacteroides fragilis-T-cell crosstalk maintains innate immune cell/pathogen homeostasis. Commensal gut Clostridia spp. suppress inflammation and induce gut tolerance. Probiotics are administered to restore immune microbiome homeostasis. Individual microbial components have an immunomodulatory effect. However, probiotic therapies for sepsis-induced immune disruptions are rarely tailored to specific immune responses. Thus, we ask the question as to how components of the intestinal microbiome, often found in probiotic therapies, affect lymphocyte phenotypic profile? Methods: T-lymphocytes were cultured with either monomicrobial or polymicrobial combinations. Microbes used were Bacteroides fragilis, Clostridium perfringens, or Lactobacillus acidophilus. Cytokines, measured by enzyme-linked immunosorbent assay (ELISA)-included interleukin (IL)-6, IL-10, IL-22, and IL-33. Flow cytometry was used for T-cell phenotyping for program-death receptor-1 (PD-1) and B- and T-lymphocyte attenuator (BTLA). T-cell DNA was extracted to assess global epigenetic changes. For translation, IL-33 was measured from surgical intensive care unit (ICU) patients with sepsis with either monomicrobial or polymicrobial infection. Results: Lactobacillus consistently induced IL-22 and IL-33. Bacteroides fragilis induced IL-33 only under polymicrobial (pB) conditions. Within surgical ICU patients, IL-33 levels were higher in polymicrobial versus monomicrobial patients. PD-1+ expression was lowest with either monomicrobial Bacteroides fragilis or Bacteroides fragilis predominant polymicrobial context. Conversely Bacteroides fragilis exposure induced a distinct PD-1-high subpopulation. B- and T-lymphocyte attenuator-positive expression did not differ after individual microbes. Among polymicrobial conditions, Bacteroides fragilis predominant (pB) and Lactobacillus acidophilus predominant (pL) increased BTLA+ expression. DNA methylation was most increased in response to Clostridium perfringens in monomicrobial and in response to Bacteroides fragilis in polymicrobial conditions. Conclusion: Unique microbe/lymphocyte interactions occur. Bacteroides fragilis induced a T-cell phenotype consistent with potential long-term immune recovery. This work begins to discover how varying microbes may induce unique functional and phenotypic T-lymphocyte responses.
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Microbioma Gastrointestinal , Microbiota , Probióticos , Sepse , Bacteroides fragilis , HumanosRESUMO
ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.
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Coinfecção/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Fatores Imunológicos/uso terapêutico , Peptídeos/uso terapêutico , Sepse/tratamento farmacológico , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Hypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic setting. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major protein phosphatase tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to development of AKI sequential hemorrhage (Hem) and cecal ligation and puncture (CLP) and whether inactivation of SHP2 through administration of its selective inhibitor, phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury. METHODS: Male C57BL/6 mice were subjected to Hem (a "priming" insult) followed by CLP or sham-Hem plus sham-CLP (S/S) as controls. Samples of blood and kidney were harvested at 24 h post CLP. The expression of neutrophil gelatinase-associated lipocalin (NGAL), high mobility group box 1 (HMGB1), caspase3 as well as SHP2:phospho-SHP2, extracellular-regulated kinase (Erk1/2): phospho-Erk1/2, and signal transducer and activator of transcription 3 (STAT3):phospho-STAT3 protein in kidney tissues were detected by Western blotting. The levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum were measured according to the manufacturer's instructions. Blood inflammatory cytokine/chemokine levels were detected by ELISA. RESULTS: We found that indices of kidney injury, including levels of BUN, Cre and NGAL as well as histopathologic changes, were significantly increased after Hem/CLP in comparison with that in the S/S group. Furthermore, Hem/CLP resulted in elevated serum levels of inflammatory cytokines/chemokines, and induced increased levels of HMGB1, SHP2:phospho-SHP2, Erk1/2:phospho-Erk1/2, and STAT3:phospho-STAT3 protein expression in the kidney. Treatment with PHPS1 markedly attenuated these Hem/CLP-induced changes. CONCLUSIONS: In conclusion, our data indicate that SHP2 inhibition attenuates AKI induced by our double-hit/sequential insult model of Hem/CLP and that this protective action may be attributable to its ability to mitigate activation of the Erk1/2 and STAT3 signaling pathway. We believe this is a potentially important finding with clinical implications warranting further investigation.
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Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Benzenossulfonatos/farmacologia , Hidrazonas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/tratamento farmacológico , Animais , Biomarcadores , Biópsia , Citocinas/metabolismo , Suscetibilidade a Doenças , Hemorragia/complicações , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Sepse/complicaçõesRESUMO
Many pathogens use the same immune evasion mechanisms as cancer cells. Patients with chronic infections have elevated levels of checkpoint receptors (e.g., programed cell death 1, PD1) on T cells. Monoclonal antibody (mAb)-based inhibitors to checkpoint receptors have also been shown to enhance T-cell responses in models of chronic infection. Therefore, inhibitors have the potential to act as a vaccine "adjuvant" by facilitating the expansion of vaccine antigen-specific T-cell repertoires. Here, we report the discovery and characterization of a peptide-based class of PD1 checkpoint inhibitors, which have a potent adaptive immunity adjuvant capability for vaccines against infectious diseases. Briefly, after identifying peptides that bind to the recombinant human PD1, we screened for in vitro efficacy in reporter assays and human peripheral blood mononuclear cells (PBMC) readouts. We first found the baseline in vivo performance of the peptides in a standard mouse oncology model that demonstrated equivalent efficacy compared to mAbs against the PD1 checkpoint. Subsequently, two strategies were used to demonstrate the utility of our peptides in infectious disease indications: (1) as a therapeutic in a bacteria-induced lethal sepsis model in which our peptides were found to increase survival with enhanced bacterial clearance and increased macrophage function; and (2) as an adjuvant in combination with a prophylactic malaria vaccine in which our peptides increased T-cell immunogenicity and the protective efficacy of the vaccine. Therefore, our peptides are promising as both a therapeutic agent and a vaccine adjuvant for infectious disease with a potentially safer and more cost-effective target product profile compared to mAbs. These findings are essential for deploying a new immunomodulatory regimen in infectious disease primary and clinical care settings.
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Doenças Transmissíveis/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Macrófagos Peritoneais/imunologia , Melanoma/imunologia , Peptídeos/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologia , Adjuvantes Imunológicos , Animais , Doenças Transmissíveis/terapia , Humanos , Células Jurkat , Melanoma Experimental , Camundongos , Biblioteca de Peptídeos , Peptídeos/síntese química , Ligação Proteica , VacinasRESUMO
This study sets out to establish the comparative contribution of PD-L1 expression by pulmonary endothelial cells (ECs) and/or epithelial cells (EpiCs) to the development of indirect acute lung injury (iALI) by taking advantage of the observation that treatment with naked siRNA by intratracheal delivery in mice primarily affects lung EpiCs, but not lung ECs, while intravenous delivery of liposomal-encapsulated siRNA largely targets vascular ECs including the lung, but not pulmonary EpiCs. We showed that using a mouse model of iALI [induced by hemorrhagic shock followed by septic challenge (Hem-CLP)], PD-L1 expression on pulmonary ECs or EpiCs was significantly upregulated in the iALI mice at 24 h post-septic insult. After documenting the selective ability of intratracheal versus intravenous delivery of PD-L1 siRNA to inhibit PD-L1 expression on EpiCs versus ECs, respectively, we observed that the iALI-induced elevation of cytokine/chemokine levels (in the bronchoalveolar lavage fluid, lung lysates, or plasma), lung myeloperoxidase and caspase-3 activities could largely only be inhibited by intravenous, but not intratracheal, delivery of PD-L1 siRNA. Moreover, intravenous, but not intratracheal, delivery led to a preservation of normal tissue architecture, lessened pulmonary edema, and reduced neutrophils influx induced by iALI. In addition, in vitro mouse endothelial cell line studies showed that PD-L1 gene knockdown by siRNA or knockout by CRISPR/Cas9-mediated gene manipulation, reduced monolayer permeability, and maintained tight junction protein levels upon recombinant IFN-γ stimulation. Together, these data imply a critical role for pulmonary vascular ECs in mediating PD-1:PD-L1-driven pathological changes resulting from systemic stimuli such as Hem-CLP.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Antígeno B7-H1/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Peroxidase/metabolismo , RNA Interferente Pequeno/metabolismo , Sepse/metabolismo , Choque Hemorrágico/metabolismoRESUMO
Therapeutic interventions to treat acute lung injury (ALI) remain largely limited to lung-protective strategies, as a real molecular pathophysiologically driven therapeutic intervention has yet to become available. While we have previously documented the expression of herpes virus entry mediator (HVEM) on leukocytes of septic mice and critically ill patients, its functional role in shock/sepsis-induced ALI has not yet been studied. Inasmuch, a murine model of indirect ALI (iALI) was induced by hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), septic challenge and HVEM-siRNA or phosphate buffered saline was administrated by intratracheal instillation 2âh after hemorrhage to determine the role of HVEM in the development of experimental iALI. Indices of lung injury were measured. HVEM expression was significantly elevated in iALI mice. Compared with phosphate buffered saline treated iALI mice, HVEM knock-down by siRNA caused a reduction of cytokine/chemokine levels, myeloperoxidase activity, broncho-alveolar lavage fluid (BALF) cell count and protein concentration. HVEM-siRNA treatment reduced inflammation and attenuated pulmonary architecture destruction as well as provided an early (60âh post HEM-CLP) survival benefit in iALI mice. This ability of anti-HVEM treatment to prevent the development of iALI and provide a transient survival benefit implies that mitigating signaling through HVEM may be a novel target worth further investigation.
Assuntos
Lesão Pulmonar Aguda/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Sepse/metabolismo , Lesão Pulmonar Aguda/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Sepse/imunologia , Internalização do VírusRESUMO
Neutrophil recruitment into the lung airspaces plays an important role in the containment and clearance of bacteria. Hemorrhagic shock, a complication of traumatic injury, induces immune dysfunction that compromises host defense and frequently leads to secondary infection. The objective of the current study was to determine whether prior hemorrhage impacts neutrophil recruitment in response to secondary Pseudomonas aeruginosa. Experiments were performed using a mouse model (C57BL/6) of respiratory infection by P. aeruginosa (strain PA103, 3â×â10 colony-forming units [CFUs]) that is delivered by intratracheal inhalation 24âh after hypovolemic hemorrhagic shock (fixed mean arterial blood pressure at 35âmmHg for 90âmin, Ringer's lactate infused as fluid resuscitation). By postmortem flow cytometry analyses of bronchoalveolar lavage fluid, we observe that prior hemorrhage attenuates the entry of neutrophils into the lung airspaces in response to P. aeruginosa. The reduction in neutrophil recruitment occurs in an amplified inflammatory environment, with elevated lung tissue levels of interleukin 6 and C-X-C motif ligand 1 in mice receiving hemorrhage prior to infection. As compared to either insult alone, outcome to sequential hemorrhage and respiratory infection includes enhanced mortality. The effect of prior hemorrhage on clearance of P. aeruginosa, as determined by quantifying bacterial CFUs in lung tissue, was not statistically significant at 24âh postinfection, but our data suggest that further inquiry may be needed to fully understand the potential impact of hemorrhagic shock on this process. These results suggest that changes in neutrophil recruitment may contribute to the immune dysfunction following hemorrhagic shock that renders the host susceptible to severe respiratory infection.
Assuntos
Hemorragia , Neutrófilos , Infecções por Pseudomonas , Pseudomonas aeruginosa/imunologia , Infecções Respiratórias , Animais , Quimiocina CXCL1/imunologia , Hemorragia/complicações , Hemorragia/imunologia , Hemorragia/patologia , Interleucina-6/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/patologia , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Infecções Respiratórias/etiologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologiaRESUMO
The liver is an organ that, when dysfunctional in a septic patient, is strongly associated with morbidity and mortality. Understanding the pathophysiology of liver failure during sepsis may lead to improved diagnostics and potential therapeutic targets. Historically, programmed cell death receptor (PD) ligand 1 (PD-L1) has been considered the primary ligand for its checkpoint molecule counterpart, PD-1, with PD-L2 rarely in the immunopathological spotlight. PD-1 and PD-L1 contribute to liver dysfunction in a murine cecal ligation and puncture (CLP) model of sepsis, but virtually nothing is known about PD-L2's role in sepsis. Therefore, our central hypothesis was that sepsis-induced changes in hepatic PD-L2 expression contributed to worsened liver function and, subsequently, more pronounced morbidity and mortality. We found that although PD-L1 gene deficiency attenuated the hepatic dysfunction seen in wild-type mice after CLP, the loss of PD-L2 appeared to actually worsen indices of liver function along with a trend toward higher liver tissue vascular permeability. Conversely, some protective effects of PD-L2 gene deletion were noted, such as reduced liver/peritoneal bacterial load and reduced IL-6, IL-10, and macrophage inflammatory protein 2 levels following CLP. These diverse actions, as well as the unique expression pattern of PD-L2, may explain why no overt survival advantage could be witnessed in the septic PD-L2-/- mice. Taken together, these data suggest that although PD-L2 has some selective effects on the hepatic response seen in the septic mouse, these factors are not sufficient to alter septic mortality in this adult murine model. NEW & NOTEWORTHY Our study shows not only that ligands of the checkpoint protein PD-1 respond inversely to a stressor such as septic challenge (PD-L2 declines, whereas PD-L1 rises) but also that aspects of liver dysfunction increase in septic mice lacking the PD-L2 gene. Furthermore, these differences in PD-L2 gene-deficient animals culminated in the abrogation of the survival advantage seen in the septic PD-L1-knockout mice, suggesting that PD-L2 may have roles beyond a simple immune tolerogen.