Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Sex-hormone-driven innate antibodies protect females and infants against EPEC infection.

Females have an overall advantage over males in resisting Gram-negative bacteremias, thus hinting at sexual dimorphism of immunity during infections. Here, through intravital microscopy, we observed a sex-biased difference in the capture of blood-borne bacteria by liver macrophages, a process that is critical for the clearance of systemic infections. Complement opsonization was indispensable for the capture of enteropathogenic Escherichia coli (EPEC) in male mice; however, a faster complement component 3-independent process involving abundant preexisting antibodies to EPEC was detected in female mice. These antibodies were elicited predominantly in female mice at puberty in response to estrogen regardless of microbiota-colonization conditions. Estrogen-driven antibodies were maternally transferrable to offspring and conferred protection during infancy. These antibodies were conserved in humans and recognized specialized oligosaccharides integrated into the bacterial lipopolysaccharide and capsule. Thus, an estrogen-driven, innate antibody-mediated immunological strategy conferred protection to females and their offspring.

A Retrospective Analysis of Leadership, Awardees, and Member Gender Representation of the Canadian Society for Immunology.

The Canadian Society for Immunology (CSI) established a formal Equity, Diversity, and Inclusion (EDI) Committee with the goal of providing EDI advocacy and leadership within the CSI, as well as in the broader scientific community. A first task of this committee was to review the publicly available historical data on gender representation within the CSI's membership, leadership, award recipients, and conference chairs/presenters as a step in establishing a baseline reference point and monitoring the trajectory of future success in achieving true inclusion. We found that, except for overall membership and a specific subset of awards, all categories showed a historical bias toward men, particularly prior to 2010. Bias persists in various categories, evident even in recent years. However, we note an encouraging trend toward greater gender parity, particularly in the roles of President, symposium presenters, and workshop chairs, especially from 2017 onward. We present these findings as well as our recommendations to enhance inclusivity. These include a more comprehensive collection and secure storage of self-identification data, emphasis on EDI as an essential component of all annual meeting activities, and innovative measures of outreach, collaboration, and leadership with the aim of making the CSI a model for improving EDI in other professional research societies.

Immune surveillance by the liver.

Receiving both portal vein blood and arterial blood, the liver is an important and critical component in the defense against blood-borne infection. To accomplish this role, the liver contains numerous innate and adaptive immune cells that specialize in detection and capture of pathogens from the blood. Further, these immune cells participate in coordinated immune responses leading to pathogen clearance, leukocyte recruitment and antigen presentation to lymphocytes within the vasculature. Finally, this role in host defense must be tightly regulated to ensure that inappropriate immune responses are not raised against nonpathogenic exogenous blood-borne molecules, such as those derived from food. It is this balance between activation and tolerance that characterizes the liver as a frontline immunological organ.

Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.

Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection.

Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: 'intrahepatic myeloid-cell aggregates for T cell population expansion') without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.

Equity, Diversity and Inclusion in Canadian immunology: communication and complexity.

The Canadian Society for Immunology (CSI) organized an Equity, Diversity and Inclusion (EDI) training workshop during its 2022 Scientific Meeting to improve understanding of EDI and explore strategies to achieve EDI goals in the scientific environment. The workshop focused on identifying Specific, Measurable, Achievable, Realistic and Timely (SMART) goals related to EDI in academia through small group discussions and learning exercises. Attendees highlighted several equity considerations within the field of academic immunology, including financial barriers, lack of diversity in research teams and gender bias; they emphasized the importance of creating an inclusive and accessible research environment. The collection and use of data relevant to EDI goals within the CSI were also identified as challenges. Fostering a culture of active and nonjudgmental listening within the CSI community is another aspirational goal to address EDI. The workshop received positive feedback from attendees, who noted that more diverse voices and specific actions for local research environments are needed.

The Lyme disease spirochete can hijack the host immune system for extravasation from the microvasculature.

Lyme disease is the most common tick-transmitted disease in the northern hemisphere and is caused by the spirochete Borrelia burgdorferi and related Borrelia species. The constellation of symptoms attributable to this malady results from vascular dissemination of B. burgdorferi throughout the body to invade various tissue types. However, little is known about the mechanism by which the spirochetes can breach the blood vessel wall to reach distant tissues. We have studied this process by direct observation of spirochetes in the microvasculature of living mice using multi-laser spinning-disk intravital microscopy. Our results show that in our experimental system, instead of phagocytizing B. burgdorferi, host neutrophils are involved in the production of specific cytokines that activate the endothelium and potentiate B. burgdorferi escape into the surrounding tissue. Spirochete escape is not induced by paracellular permeability and appears to occur via a transcellular pathway. Neutrophil repurposing to promote bacterial extravasation represents a new and innovative pathogenic strategy.

Acetylsalicylic acid inhibits intravascular coagulation during Staphylococcus aureus-induced sepsis in mice.

Antiplatelet therapies have been proposed for the treatment of sepsis, a syndrome resulting from a dysregulated immune response and inappropriate activation of coagulation. Acetylsalicylic acid (ASA) may serve as a potential therapeutic strategy to prevent infection-induced coagulopathy and associated tissue damage. Using intravital microscopy, we found that Staphylococcus aureus infection induced neutrophil recruitment, platelet aggregation, and neutrophil extracellular trap (NET) release in the liver. Mice pretreated with ASA, or animals receiving ASA 3 hours postinfection, had significantly reduced platelet aggregation and NET release. Additionally, ASA-treated mice had reduced intravascular thrombin activity and microvascular occlusion as compared with untreated S aureus-infected mice. This inhibition of coagulation was accompanied by decreased levels of alanine aminotransferase and aspartate aminotransferase in the plasma, indicating less liver damage. Finally, bacterial loads (colony-forming units per milliliter) in liver, lung, and spleen were not different between groups, and the phagocytic capacity of Kupffer cells was preserved following ASA treatment. These results suggest that ASA may serve as a therapeutic approach to sepsis through its ability to reduce the deleterious action of immunothrombi while maintaining innate immune functions.

Exploratory Evaluation of the Relationship Between iNKT Cells and Systemic Cytokine Profiles of Critically Ill Patients with Neurological Injury.

BACKGROUND: Neurological injury can alter the systemic immune system, modifying the functional capacity of immune cells and causing a dysfunctional balance of cytokines, although mechanisms remain incompletely understood. The objective of this study was to assess the temporal relationship between changes in the activation status of circulating invariant natural killer T (iNKT) cells and the balance of plasma cytokines among critically ill patients with neurological injury. METHODS: We conducted an exploratory prospective observational study of adult (18 years or older) intensive care unit (ICU) patients with acute neurological injury (n = 20) compared with ICU patients without neurological injury (n = 22) and healthy controls (n = 10). Blood samples were collected on days 1, 2, 4, 7, 14, and 28 following ICU admission to analyze the activation status of circulating iNKT cells by flow cytometry and the plasma concentration of inflammation-relevant immune mediators, including T helper 1 (TH1) and T helper 2 (TH2) cytokines, by multiplex bead-based assay. RESULTS: Invariant natural killer T cells were activated in both ICU patient groups compared with healthy controls. Neurological patients had decreased levels of multiple immune mediators, including TH1 cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-12p70), indicative of immunosuppression. This led to a greater than twofold increase in the ratio of TH2/TH1 cytokines early after injury (days 1 - 2) compared with healthy controls, a shift that was also observed for ICU controls. Systemic TH2/TH1 cytokine ratios were positively associated with iNKT cell activation in the neurological patients and negatively associated in ICU controls. These relationships were strongest for the CD4+ iNKT cell subset compared with the CD4- iNKT cell subset. The relationships to individual cytokines similarly differed between patient groups. Forty percent of the neurological patients developed an infection; however, differences for the infection subgroup were not identified. CONCLUSIONS: Critically ill patients with neurological injury demonstrated altered systemic immune profiles early after injury, with an association between activated peripheral iNKT cells and elevated systemic TH2/TH1 cytokine ratios. This work provides further support for a brain-immune axis and the ability of neurological injury to have far-reaching effects on the body's immune system.

Netting Liver Disease: Neutrophil Extracellular Traps in the Initiation and Exacerbation of Liver Pathology.

The liver plays a vital role in the immune system. Its unique position in the portal circulation and the architecture of the hepatic sinusoids, in combination with the wide-ranged population of immunocompetent cells, make the liver function as an immune filter. To aid in pathogen clearance, once challenged, the liver initiates the rapid recruitment of a wide variety of inflammatory cells, including neutrophils. These neutrophils, in conjunction with platelets, facilitate the release of neutrophil extracellular traps (NETs), which are web-like structures of decondensed nuclear DNA, histones, and neutrophil proteins. NETs function as both a physical and a chemical barrier, binding and killing pathogens circulating in the blood stream. In addition to their antimicrobial role, NETs also bind platelets, activate coagulation, and exacerbate host inflammatory response. This interplay between inflammation and coagulation drives microvascular occlusion, ischemia, and (sterile) damage in liver disease. Although direct clinical evidence of this interplay is scarce, preliminary studies indicate that NETs contribute to progression of liver disease and (thrombotic) complications. Here, we provide an overview of the pathological mechanisms of NETs in liver disease. In addition, we summarize clinical evidence for NETs in different disease etiologies and complications of liver disease and discuss the possible implications for the use of NETs as a diagnostic marker and a therapeutic target in liver disease.

The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse strain and in a patient with severe combined immunodeficiency.

Mice carrying the recessive locus for peripheral T cell deficiency (Ptcd) have a block in thymic egress, but the mechanism responsible is undefined. Here we found that Ptcd T cells had an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus showed a point substitution of lysine for glutamic acid at position 26 in the actin regulator coronin 1A that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. The discovery of another coronin 1A mutant during an N-ethyl-N-nitrosourea-mutagenesis screen for T cell-lymphopenic mice prompted us to evaluate a T cell-deficient, B cell-sufficient and natural killer cell-sufficient patient with severe combined immunodeficiency, whom we found had mutations in both CORO1A alleles. Our findings establish a function for coronin 1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation and demonstrate that actin regulation is a biological process defective in human and mouse severe combined immunodeficiency.

Inhibition of immunothrombosis does not affect pathogen capture and does not promote bacterial dissemination in a mouse model of sepsis.

After infection, neutrophils release neutrophil extracellular traps (NETs), decondensed DNA fibers decorated with both nuclear proteins and proteins derived from intracellular granules. These structures have a fundamental role in the development of immunothrombosis; a physiological process mediated by immune cells and molecules from the coagulation system that facilitates the recognition, containment, and destruction of pathogens. Although NETs and immunothrombi are widely hypothesized to be key host defense responses responsible for limiting bacterial dissemination, their actual role in this process has not been formally assessed within the context of a bloodstream infection. Mice were first treated with LPS to generate inflammation (NETs and immunothrombi) and then bacteria dissemination was analyzed by intravital microscopy and colony-forming units (CFU) assay. Blocking NETs or coagulation by the administration of DNase or Argatroban (thrombin inhibitor), respectively, did not modify the percentage of bacteria capture by Kupffer cells, neutrophils or platelets. Moreover, both inhibitors reduced the number of bacteria in the spleen, without modifying CFUs in the liver or lung. In conclusion, we demonstrate that immunothrombi are not necessary to limit the dissemination of bloodstream bacterial infections.

Role of platelets in neutrophil extracellular trap (NET) production and tissue injury.

In addition to their well-known role as the cellular mediator of thrombosis, numerous studies have identified key roles for platelets during various disease processes. Importantly, platelets play a critical role in the host immune response, directly interacting with, and eliminating pathogens, from the blood stream. In addition to pathogen clearance, platelets also contribute to leukocyte recruitment at sites of infection and inflammation, and modulate leukocyte activity. Platelet interaction with activated neutrophils is a potent inducer of neutrophil extracellular trap (NET). NETs consist of a diffuse, sticky web of extracellular DNA, nuclear and granular proteins, and serve to ensnare and kill pathogens. In addition to catching pathogens, the cytotoxic molecules and proteases on NETs have the potential to inflict significant tissue damage. Additionally, NET components have been suggested to be key activators of infection-induced coagulopathy. These critical roles, at the interface between hemostasis and immunity, highlight the need for balance in the platelet response; too little platelet activity results in bleeding and immune deficit, too much leads to tissue pathogenesis. In this review, we highlight recent advances in our understanding of the role platelets play in inflammation, the link between platelets and NETs and the role platelets play in disease pathogenesis.

Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice.

Neutrophil extracellular traps (NETs; webs of DNA coated in antimicrobial proteins) are released into the vasculature during sepsis where they contribute to host defense, but also cause tissue damage and organ dysfunction. Various components of NETs have also been implicated as activators of coagulation. Using multicolor confocal intravital microscopy in mouse models of sepsis, we observed profound platelet aggregation, thrombin activation, and fibrin clot formation within (and downstream of) NETs in vivo. NETs were critical for the development of sepsis-induced intravascular coagulation regardless of the inciting bacterial stimulus (gram-negative, gram-positive, or bacterial products). Removal of NETs via DNase infusion, or in peptidylarginine deiminase-4-deficient mice (which have impaired NET production), resulted in significantly lower quantities of intravascular thrombin activity, reduced platelet aggregation, and improved microvascular perfusion. NET-induced intravascular coagulation was dependent on a collaborative interaction between histone H4 in NETs, platelets, and the release of inorganic polyphosphate. Real-time perfusion imaging revealed markedly improved microvascular perfusion in response to the blockade of NET-induced coagulation, which correlated with reduced markers of systemic intravascular coagulation and end-organ damage in septic mice. Together, these data demonstrate, for the first time in an in vivo model of infection, a dynamic NET-platelet-thrombin axis that promotes intravascular coagulation and microvascular dysfunction in sepsis.

Cytokines and Chemokines in Pediatric Appendicitis: A Multiplex Analysis of Inflammatory Protein Mediators.

OBJECTIVES: We aimed to demonstrate the potential of precision medicine to describe the inflammatory landscape present in children with suspected appendicitis. Our primary objective was to determine levels of seven inflammatory protein mediators previously associated with intra-abdominal inflammation (C-reactive protein-CRP, procalcitonin-PCT, interleukin-6 (IL), IL-8, IL-10, monocyte chemoattractant protein-1-MCP-1, and serum amyloid A-SAA) in a cohort of children with suspected appendicitis. Subsequently, using a multiplex proteomics approach, we examined an expansive array of novel candidate cytokine and chemokines within this population. METHODS: We performed a secondary analysis of targeted proteomics data from Alberta Sepsis Network studies. Plasma mediator levels, analyzed by Luminex multiplex assays, were evaluated in children aged 5-17 years with nonappendicitis abdominal pain (NAAP), acute appendicitis (AA), and nonappendicitis sepsis (NAS). We used multivariate regression analysis to evaluate the seven target proteins, followed by decision tree and heat mapping analyses for all proteins evaluated. RESULTS: 185 children were included: 83 with NAAP, 79 AA, and 23 NAS. Plasma levels of IL-6, CRP, MCP-1, PCT, and SAA were significantly different in children with AA compared to those with NAAP (p < 0.001). Expansive proteomic analysis demonstrated 6 patterns in inflammatory mediator profiles based on severity of illness. A decision tree incorporating the proteins CRP, ferritin, SAA, regulated on activation normal T-cell expressed and secreted (RANTES), monokine induced by gamma interferon (MIG), and PCT demonstrated excellent specificity (0.920) and negative predictive value (0.882) for children with appendicitis. CONCLUSIONS: Multiplex proteomic analyses described the inflammatory landscape of children presenting to the ED with suspected appendicitis. We have demonstrated the feasibility of this approach to identify potential novel candidate cytokines/chemokine patterns associated with a specific illness (appendicitis) amongst those with a broad ED presentation (abdominal pain). This approach can be modelled for future research initiatives in pediatric emergency medicine.

Macrophage Uptake of Necrotic Cell DNA Activates the AIM2 Inflammasome to Regulate a Proinflammatory Phenotype in CKD.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1ß cleavage in Aim2-/- or WT mice that received WT bone marrow than in WT mice that received Aim2-/- bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5-6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1ß, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1ß levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

Platelets as Modulators of Inflammation.

Platelets have classically been considered crucial effector cells in hemostasis, but now are increasingly recognized as players during inflammatory responses in innate and adaptive immunity. Platelets can recognize and kill invading pathogens, and, upon stimulation, also release a wide array of mediators that modify immune and endothelial cell responses. Increased platelet activity can protect the host against infectious insults; however, the excessive activity can lead to inflammation-mediated tissue damage. These critical roles highlight the necessity of balancing the platelet response at the intersection of hemostasis and inflammation. In this review, the authors present the current understanding of the inflammatory role of platelets. They also highlight recent findings on a modulator that links inflammation and deleterious tissue damage in disease pathogenesis.

Neutrophils in viral infection.

Neutrophils are the first wave of recruited immune cells to sites of injury or infection and are crucial players in controlling bacterial and fungal infections. Although the role of neutrophils during bacterial or fungal infections is well understood, their impact on antiviral immunity is much less studied. Furthermore, neutrophil function in tumor pathogenesis and cancer treatment has recently received much attention, particularly within the context of oncolytic virus infection where neutrophils produce antitumor cytokines and enhance oncolysis. In this review, multiple functions of neutrophils in viral infections and immunity are discussed. Understanding the role of neutrophils during viral infection may provide insight into the pathogenesis of virus infections and the outcome of virus-based therapies.

Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx. METHODS: We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry. RESULTS: We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopaenia was induced 2 hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor (VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. CONCLUSIONS: A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration.