Pyroglutamate-3 Amyloid-β Deposition in the Brains of Humans,Non-Human Primates,Canines, and Alzheimer Disease–Like Transgenic Mouse Models

Amyloid-β (Aβ) peptides, starting with pyroglutamate at the third residue (pyroGlu-3 Aβ), are a major species deposited in the brain of Alzheimer disease (AD) patients. Recent studies suggest that this isoform shows higher toxicity and amyloidogenecity when compared to full-length Aβ peptides. Here, we report the first comprehensive and comparative IHC evaluation of pyroGlu-3 Aβ deposition in humans and animal models. PyroGlu-3 Aβ immunoreactivity (IR) is abundant in plaques and cerebral amyloid angiopathy of AD and Down syndrome patients, colocalizing with general Aβ IR. PyroGlu-3 Aβ is further present in two nontransgenic mammalian models of cerebral amyloidosis, Caribbean vervets, and beagle canines. In addition, pyroGlu-3 Aβ deposition was analyzed in 12 different AD-like transgenic mouse models. In contrast to humans, all transgenic models showed general Aβ deposition preceding pyroGlu-3 Aβ deposition. The findings varied greatly among the mouse models concerning age of onset and cortical brain region. In summary, pyroGlu-3 Aβ is a major species of β-amyloid deposited early in diffuse and focal plaques and cerebral amyloid angiopathy in humans and nonhuman primates, whereas it is deposited later in a subset of focal and vascular amyloid in AD-like transgenic mouse models. Given the proposed decisive role of pyroGlu-3 Aβ peptides for the development of human AD pathology, this study provides insights into the usage of animal models in AD studies.Alzheimer disease (AD) is the most common form of dementia, predicted to affect approximately 42 million people worldwide in the year 2020.¹ The two prominent histopathological hallmarks of AD are extracellular neuritic plaques composed of aggregated amyloid-β protein (Aβ) and intracellular neurofibrillary tangles comprising hyperphosphorylated tau.^2,3 Aβ is formed via the amyloidogenic pathway in which the amyloid precursor protein (APP) is liberated by two sequential endopeptidase cleavages (β- and γ-secretase).⁴ Besides a marked C-terminal heterogeneity of Aβ peptides represented by the isoforms Aβ40 and Aβ42, N-terminal variants are also frequently found, eg, pyroGlu-3 Aβ and pyroGlu-11 Aβ.⁵N-terminally truncated and modified Aβ species have been shown to be a major component of Aβ deposited in plaques and vessels of AD and Down syndrome (DS) patients.^6–9 Current hypotheses suggest that pyroGlu-3 Aβ may play an early and seminal role in the oligomerization and seeding of Aβ in familial AD (FAD) and sporadic AD.^10–12 PyroGlu-3 Aβ is formed by cyclization of glutamate residues 3 or 11 by glutaminyl cyclase (QC).¹³ An N-terminal truncation of Aβ precedes formation of pyroglutamic acid. Such post-translationally modified species have been shown to be highly toxic to neuronal and glial cultures.¹⁴ When compared to unmodified Aβ, pyroGlu-3 Aβ has a higher aggregation propensity and stability, and exhibits increased potential to interfere with hippocampal LTP.^15–17 Inhibition of QC has been shown to prevent pyroGlu-3 Aβ formation in vitro¹⁸ and in vivo.^19,20 Accordingly, QC inhibitors are currently in development as a novel pharmacological approach to treat and/or prevent AD.²⁰With the advent of numerous preclinical models, it is now possible to mimic at least some of the pathological hallmarks of AD for characterization and testing of potential treatments. With regard to the established role of pyroGlu-3 Aβ in AD pathology represented by neuron loss and cognitive decline,^12,21,22 a detailed characterization of pyroGlu-3 Aβ distribution of available transgenic (tg) and nontransgenic models and their comparison to human pathology is urgently needed. In this regard, recent research has focused only on select models.^23–26Here, we completed detailed IHC analyses in human AD, nondemented aged controls (AC), and DS brains, as well as in the Caribbean vervet,²⁷ aged beagle canines,²⁸ and 12 AD-like tg mouse strains. Our results support the early deposition of pyroGlu-3 Aβ along with general Aβ in humans, nonhuman primates, and canines, in contrast to later deposition in AD-like tg mouse models, underlining differences in plaque pathology between nontransgenic/nonrodent and transgenic murine model systems.     

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.A large body of evidence indicates that soluble oligomers of amyloid-β (Aβ) are the primary toxic peptides that initiate downstream tau pathology in the amyloid cascade hypothesis of Alzheimer disease (AD).^{1, 2} However, the time course and severity of AD dementia have been generally found to correlate with neurofibrillary tangle development rather than plaque appearance,^{3, 4, 5, 6, 7, 8} although a few studies have linked plaques with early cognitive decline.^{9, 10, 11, 12} Soluble oligomeric Aβ has been highlighted as the primary toxin for loss of dendritic spines and synaptic function¹³ and has also been directly linked to downstream tau pathology. For example, suppression of a tau kinase pathway can prevent Aβ42 oligomer-induced dendritic spine loss,¹⁴ and injection of Aβ42 fibrils into mutant tau mice induces neurofibrillary tangles in cell bodies retrograde to the injections.¹⁵ In vivo, effects of Aβ oligomers versus fibrils are harder to separate; however, lowering soluble Aβ oligomers by halving β–site amyloid precursor protein (APP) cleaving enzyme reduces accumulation and phosphorylation of wild-type tau in a mouse model.¹⁶ Evidence for Aβ and tau association is particularly strong in the dendritic compartment, where tau was shown to mediate Aβ toxicity via linkage of fyn to downstream N-methyl-d-aspartate receptor toxicity.¹⁷The earliest cognitive losses in AD have long been thought to correlate with synapse loss.^{8, 18, 19, 20, 21} In humans, electron microscopic studies have documented synapse-associated Aβ and tau,^{22, 23} and much work documents activity-dependent release of synaptic Aβ into interstitial fluid, which drives local Aβ deposition in human subjects and in rodents.^{4, 24, 25} Of importance, most synapse-associated Aβ in cortical synapses of AD patients consists of soluble oligomeric species,²⁶ and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers.^{27, 28, 29, 30, 31} With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Aβ and hyperphosphorylated tau (p-tau) pathology in synaptic terminals. In addition to early- and late-stage disease, the AD samples included nondemented high pathology controls (HPCs) with substantial AD-related pathology. Synaptic accumulation of Aβ occurred in the earliest plaque stages, before the appearance of synaptic p-tau, which did not appear until late-stage disease. Soluble Aβ oligomers in synaptic terminals were elevated in early AD cases compared with HPCs, indicating an association with the onset of a dementia diagnosis.     

MyD88-Dependent Signaling Prolongs Survival and Reduces Bacterial Burden during Pulmonary Infection with Virulent Francisella tularensis

Francisella tularensis is the causative agent of the debilitating febrile illness tularemia. The severe morbidity associated with F. tularensis infections is attributed to its ability to evade the host immune response. Innate immune activation is undetectable until more than 48 hours after infection. The ensuing inflammatory response is considered pathological, eliciting a septic-like state characterized by hypercytokinemia and cell death. To investigate potential pathological consequences of the innate immune response, mice deficient in a key innate immune signaling molecule, MyD88, were studied. MyD88 knockout (KO) mice were infected with the prototypical virulent F. tularensis strain, Schu S4. MyD88 KO mice succumbed to infection more rapidly than wild-type mice. The enhanced pathogenicity of Schu S4 in MyD88 KO mice was associated with greater bacterial burdens in lungs and distal organs, and the absence of IFN-γ in the lungs, spleens, and sera. Cellular infiltrates were not observed on histological evaluation of the lungs, livers, or spleens of MyD88 KO mice, the first KO mouse described with this phenotype to our knowledge. Despite the absence of cellular infiltration, there was more cell death in the lungs of MyD88 KO mice. Thus, the host proinflammatory response is beneficial, and MyD88 signaling is required to limit bacterial burden and prolong survival during pulmonary infection by virulent F. tularensis.Inhalation of Francisella tularensis results in pneumonic tularemia, which is the most severe form of disease and associated with mortality rates up to 30% to 60% among untreated individuals.^1,2 Additionally, its low infectious dose of <10 colony-forming units (CFU) and high morbidity have led to its incorporation into previous governmental bioweapons programs and prompted the Centers for Disease Control and Prevention to list F. tularensis as a Tier 1 select agent.^1–3 There are two clinically relevant subspecies of F. tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B), with the former being the most infectious and causing the most severe form of disease.⁴ Due to the restrictive biocontainment needed to work with these strains, attenuated strains such as F. tularensis subsp. holarctica Live Vaccine Strain (LVS) are often used to model the more virulent strains of F. tularensis.F. tularensis is considered to be a stealth pathogen due to its ability to evade immune detection.⁵ Following pulmonary exposure, F. tularensis replicates within macrophages and dendritic cells while simultaneously suppressing their activation and thereby limiting the production of proinflammatory cytokines.^6–9 Robust bacterial replication and dissemination to distal organs occur during the early stages of infection while there is not a detectable host response.¹⁰ The lack of an innate immune response early during infection is important for the extreme virulence of F. tularensis. Administration of nontypeable Haemophilus influenzae or acai berry polysaccharide before infection with F. tularensis activates the innate immune response and prolongs survival of mice.^11,12 Additionally, mutants of F. tularensis that stimulate the host immune response more robustly are less virulent in a pulmonary model of tularemia.¹³ Taken together, F. tularensis is able to evade the host immune response for at least 48 hours after infection. However, early induction of the host response decreases the morbidity of mice infected with F. tularensis, suggesting that the bacterium is susceptible to an activated host response.Cytokines and recruited immune cells are readily detected in the lungs by 72 hours after infection, but it is uncertain whether this host response is beneficial or detrimental.^6,10,14 The immune response has been characterized as a septic-like state with hypercytokinemia in the organs and blood, bacteremia, and depletion of immune cells.^15–17 To control LVS infection, a clear role has been established for a variety of cell populations, cytokines, and signaling pathways of the innate immune response, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), Toll-like receptor 2 (TLR2), and myeloid differentiation primary response 88 (MyD88).^18,19 However, direct assessments of neutrophils, natural killer cells, IFN-γ, and TNF-α during primary tularemia with virulent F. tularensis strains suggest they do not have a comparable role.^14,20,21 Production of host matrix metalloprotease 9, which is important for the recruitment of neutrophils, also exacerbates the pathogenesis of F. tularensis.²² However, the septic-like state also occurs in mice infected with attenuated Francisella strains at nonlethal doses of bacteria,¹⁴ suggesting that the correlation with death is not causal. There is also a plateau in the bacterial burden subsequent to immune activation, consistent with the host response restricting bacterial growth.¹⁴ Using a convalescent model in which antibiotic administration extends the life of the mice infected with F. tularensis, beneficial contributions of IFN-γ, IL-12, T cells, and B cells were established that could not otherwise be observed in a native infection.²³ Investigations of the convalescent model suggest that there is a role for the immune response in combating virulent strains of F. tularensis, which contrasts with previous studies investigating the cytokines and cell populations during F. tularensis infection in naive mice.²¹ Therefore, additional studies investigating the innate host response against virulent F. tularensis are required to clarify the role for this response and to define its contribution to F. tularensis pathogenesis.The innate immune response in conjunction with the epithelial barrier provides the first line of defense against pathogens. The Toll/IL-1R–receptor (TIR) domain–containing proteins play a crucial role in innate immune responses.²⁴ The TIR family comprises TLRs and IL-1 family receptors, as well as adaptor proteins required for signal transduction from the receptors to induce proinflammatory cytokines.²⁵ MyD88 is a TIR domain–containing adaptor protein for signaling from IL-1 family receptors and all TLRs except for TLR3, which makes MyD88 an important signaling molecule of the initial host response.²⁶ For many bacterial pathogens, the host requires MyD88 signaling to successfully control and clear the infection. MyD88-dependent signaling is often required to prolong survival,^27–30 for cellular recruitment and inflammation,^27,29–31 and for restriction of bacterial burden.^28,31 In circumstances where MyD88 signaling is required, it is almost universally needed for cytokine and chemokine responses.^{27–29,31–33} By contrast, there is at least one scenario where MyD88 signaling is actually detrimental to the host response.³⁴ Thus, although MyD88 may be important generally, the phenotypic requirement for MyD88 is specific for each pathogen.The nearly ubiquitous need for MyD88 signaling during the innate host response to microbial infections makes it a good model to explore the contribution of host activation to F. tularensis pathogenesis. Microbiological, immunological, and natural history outcomes were assessed by comparing wild-type (WT) and MyD88 KO mice infected with the prototypical type A strain of F. tularensis, Schu S4, to identify the possible beneficial and detrimental roles of the innate host response. MyD88 KO mice had reduced survival, greater bacterial burdens, and more cell death in the lungs. Collectively, these data demonstrate a beneficial role for the innate immune response during primary pneumonic tularemia with virulent F. tularensis.     

Suppressed Accumulation of Cerebral Amyloid β Peptides in Aged Transgenic Alzheimer’s Disease Mice by Transplantation with Wild-Type or Prostaglandin E2 Receptor Subtype 2-Null Bone Marrow

A complex therapeutic challenge for Alzheimer’s disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid β (Aβ) peptides. One potential target is selective suppression of microglial prostaglandin E₂ receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2^−/−) into lethally irradiated 5-month-old wild-type or APPswe-PS1ΔE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2^−/− microglia was more efficient in APPswe-PS1ΔE9 than in wild-type mice, and APPswe-PS1ΔE9 mice that received EP2^−/− BMT had increased cortical microglia compared with APPswe-PS1ΔE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Aβ plaques and potentially more neurotoxic soluble Aβ species. An additional 25% reduction in cerebral cortical Aβ burden was achieved in mice that received EP2^−/− BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Aβ peptide and plaque accumulation in the cerebrum of patients with AD.Alzheimer’s disease (AD), the most common dementing neurodegenerative disease,¹ is a major public health burden for older Americans.² Amyloid β (Aβ) peptides are pleotropic molecules that are directly neurotoxic and stimulate liberation of cytotoxic cytokines through activation of microglia innate immune response.³ However, activated microglia phagocytosis and degradation of Aβ species is key to cerebral Aβ homeostasis.⁴ Thus, an important but complex therapeutic challenge is balancing deleterious and beneficial aspects of microglial activation in AD.⁵ One proposed mechanism of microglial modulation is prostaglandin E₂ signaling, especially through activation of the E prostanoid receptor subtype 2 (EP2).⁶ Cultured microglia lacking EP2 (EP2^−/−) show enhanced phagocytosis of Aβ from human brain explants and reduced paracrine neurotoxicity.⁷ In vivo experiments with EP2^−/− mice have shown reduced accumulation of cerebral Aβ in a transgenic mouse model of AD,^7,8,9 as well as suppressed oxidative damage to neurons following innate immune activation.^7,10,11,12 However, because EP2 is expressed by several cell types in brain, including microglia and neurons, the importance of microglial-specific EP2 has not been established. To address this gap in our knowledge, bone marrow cells from EP2^−/− mice were transplanted into APPswe-PS1ΔE9 mice.Circulating bone marrow transplant (BMT)-derived cells can selectively replace resident microglia,¹³ and up to 30% of microglia can be derived from donor marrow in wild-type mice recipients up to a year after transplantation.^14,15 Moreover, engraftment of brain appears qualitatively more efficient in recipient AD mice than in wild-type controls.^16,17 The reasons for the apparent higher engraftment are not clear, but may be in response to chronic low level immune activation in AD mouse brains.^16,17 Some investigators have shown BMT-derived microglia associated with Aβ deposits in vivo, and that transgenic AD mouse BMT recipients have reduced Aβ plaque burden.¹⁷ Although previous data addressed potential mechanisms by which BMT-derived microglia might promote clearance of Aβ peptides,¹⁸ the results of these studies were confounded by the effects of preconditioning brain irradiation; it is possible that the reduced Aβ plaque burden was caused by irradiation-induced alteration of Aβ production or clearance rather than BMT-derived microglia. In the current studies, we robustly quantify microglial engraftment in brains of APPswe-PS1ΔE9 mice. In addition, we control for the potential confounder of irradiation-mediated Aβ peptide suppression by evaluating Aβ in mice that received cranial-specific irradiation with or without BMT. Finally, we test the hypothesis that BMT with cells from EP2^−/− mice would enhance cerebral bone marrow derived microglia engraftment and clearance of Aβ peptides from cerebrum of APPswe-PS1ΔE9 mice.     

The Involvement of Collagen Triple Helix Repeat Containing 1 in Muscular Dystrophies

Apolipoprotein E4 (APOE4) genotype is the strongest genetic risk factor for late-onset Alzheimer disease and confers a proinflammatory, neurotoxic phenotype to microglia. Here, we tested the hypothesis that bone marrow cell APOE genotype modulates pathological progression in experimental Alzheimer disease. We performed bone marrow transplants (BMT) from green fluorescent protein–expressing human APOE3/3 or APOE4/4 donor mice into lethally irradiated 5-month-old APPswe/PS1ΔE9 mice. Eight months later, APOE4/4 BMT–recipient APPswe/PS1ΔE9 mice had significantly impaired spatial working memory and increased detergent-soluble and plaque Aβ compared with APOE3/3 BMT–recipient APPswe/PS1ΔE9 mice. BMT-derived microglia engraftment was significantly reduced in APOE4/4 recipients, who also had correspondingly less cerebral apoE. Gene expression analysis in cerebral cortex of APOE3/3 BMT recipients showed reduced expression of tumor necrosis factor-α and macrophage migration inhibitory factor (both neurotoxic cytokines) and elevated immunomodulatory IL-10 expression in APOE3/3 recipients compared with those that received APOE4/4 bone marrow. This was not due to detectable APOE-specific differences in expression of microglial major histocompatibility complex class II, C-C chemokine receptor (CCR) type 1, CCR2, CX3C chemokine receptor 1 (CX3CR1), or C5a anaphylatoxin chemotactic receptor (C5aR). Together, these findings suggest that BMT-derived APOE3-expressing cells are superior to those that express APOE4 in their ability to mitigate the behavioral and neuropathological changes in experimental Alzheimer disease.Humans uniquely have three different apolipoprotein E (APOE) alleles (ɛ2, ɛ3, and ɛ4). APOE4 is the single greatest genetic risk factor for late-onset Alzheimer disease (AD), and there is a gene dosage effect.¹ However, genetic association does not inform function/pathogenesis. Multiple mechanisms have been postulated that predominantly focus on production, metabolism, or clearance of amyloid-β (Aβ) and that are variably supported by multiple observations, including: i) APOE genotype is strongly related to Aβ levels in brain and cerebrospinal fluid of AD patients^2,3; ii) modulation of apolipoprotein E (apoE) protein levels in brain results in alterations of Aβ burden^4,5; iii) Aβ degradation is at least partially apoE dependent^6,7; and iv) Aβ clearance is differentially modulated by apoE isoforms, with APOE4 mice exhibiting reduced central and peripheral Aβ clearance compared with APOE3 mice.^8–10 Aβ degradation and clearance is at least partially dependent on microglia, the innate immune effector cells of the brain. Microglia have migratory and phagocytic capacity, are increased in the vicinity of Aβ plaques, and phagocytose Aβ.^11–13 APOE genotype modulates central nervous system innate immune function in culture,¹⁴ including astrocyte and microglia elaboration of cytokines and chemokines,^15,16 microglia production of reactive oxygen species,¹⁷ microglia-mediated paracrine neurotoxicity,¹⁸ microglia migration,¹⁹ and other functions.²⁰ However, the specific contribution of microglial APOE genotype to AD pathophysiology in vivo is largely unknown.To address this critical question and to test a potential therapeutic application, we used the fact that bone marrow transplantation (BMT) results in the gradual replacement of endogenous (host) microglia (to the near exclusion of other cell types) with microglia derived from donor marrow, in both wild-type mice and transgenic mouse models of AD.^21–24 We used targeted-replacement (TR) APOE mice homozygous for either the APOE3 or APOE4 gene inserted into the mouse APOE regulatory elements^25,26 that coexpressed green fluorescent protein (GFP). We transplanted whole bone marrow (BM) isolated from TR APOE3/3;GFP or TR APOE4/4;GFP mice into lethally irradiated APPswe/PS1ΔE9 mice to determine the specific role of microglial APOE genotype in the pathological progression of AD.     

Aβ Accelerates the Spatiotemporal Progression of Tau Pathology and Augments Tau Amyloidosis in an Alzheimer Mouse Model

Senile plaques formed by β-amyloid peptides (Aβ) and neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau, a microtubule-associated protein, are the hallmark lesions of Alzheimer''s disease (AD) in addition to loss of neurons. While several transgenic (Tg) mouse models have recapitulated aspects of AD-like Aβ and tau pathologies, a spatiotemporal mapping paradigm for progressive NFT accumulation is urgently needed to stage disease progression in AD mouse models. Braak and co-workers developed an effective and widely used NFT staging paradigm for human AD brains. The creation of a Braak-like spatiotemporal staging scheme for tau pathology in mouse models would facilitate mechanistic studies of AD-like tau pathology. Such a scheme would also enhance the reproducibility of preclinical AD therapeutic studies. Thus, we developed a novel murine model of Aβ and tau pathologies and devised a spatiotemporal scheme to stage the emergence and accumulation of NFTs with advancing age. Notably, the development of NFTs followed a spatiotemporal Braak-like pattern similar to that observed in authentic AD. More significantly, the presence of Aβ accelerated NFT formation and enhanced tau amyloidosis; however, tau pathology did not have the same effect on Aβ pathology. This novel NFT staging scheme provides new insights into the mechanisms of tau pathobiology, and we speculate that this scheme will prove useful for other basic and translational studies of AD mouse models.Alzheimer''s disease (AD) is characterized by a triad of neuropathological hallmarks including senile plaques, neurofibrillary tangles (NFTs), and neuron loss. Senile plaques are extracellular lesions composed of β-amyloid (Aβ) peptides, whereas NFTs are composed mainly of hyperphosphorylated tau, a microtubule-associated protein. Previous reports using the six-stage NFT progression scheme developed by Braak and co-workers defined a spatiotemporal pattern of tangle accumulation which correlates more closely with the severity of dementia in AD patients than the burden of Aβ plaques.^1–3 Despite the fact that the burden of Aβ plaques correlates less well with the degree of dementia, the amyloid cascade hypothesis posits that Aβ influences NFT evolution, although other evidence suggests tangles precede plaque formation.^4–6 Thus, the influence of plaques and tangles on each other remains controversial, and methodological limitations of postmortem studies of AD brains precludes unequivocal resolution of this controversy.Thus, to probe the interplay between plaques and tangles, several transgenic (Tg) mouse models with both plaque and tangle pathology have been described as reviewed elsewhere.⁷ For example, bigenic mouse models harboring both human mutant tau and APP transgenes display enhanced tau tangle formation when compared to their monogenic counterparts.^8–10 These results lend support to the view that plaques influence the development of tangles by augmenting NFT formation. However, the lack of a rigorous strategy to define the spatiotemporal accumulation of NFTs limits clear understanding of the impact plaques exert on progressive tau pathology. Moreover, the availability of a tangle staging scheme would facilitate comparative studies of AD pathology across different Tg mice as well as enable more standardized and rigorous assessment of the effects of potential disease-modifying therapies. The need for standardization of Tg mouse studies was also emphasized in a recent assessment of the failure to reproduce many preclinical studies in Tg mouse models of amyotrophic lateral sclerosis.¹¹ Hence, we addressed this issue by generating a novel bigenic mouse model of Aβ and tau pathology through crossing our P301S mutant tau (PS19) Tg mouse model with the well-known PDAPP model that overexpresses mutant V717F APP.^12,13 Using this PS19;PDAPP bigenic model, we staged the spatiotemporal pattern of progressive accumulations of tau pathology and showed an accumulation similar to the Braak NFT staging scheme for AD brains. We then exploited this staging scheme to show that the presence of Aβ alters the progressive accumulation of tau pathology. Thus, by developing a tau staging scheme, we provide insights into the modulation of tau pathology by Aβ. We speculate that our novel staging paradigm will lead to additional insights into mechanisms of AD and prove useful in other basic and translational studies of AD mouse models.     

Homozygosity and Heterozygosity for Null Col5a2 Alleles Produce Embryonic Lethality and a Novel Classic Ehlers-Danlos Syndrome–Related Phenotype

Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2^−/− homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2^−/− embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2^−/− and Col5a1^−/− fibril formation and embryonic survival suggest that α1(V)₃ homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2^+/− adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2^+/− adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2^+/− humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions.Collagen V is a low-abundance fibrillar collagen widely distributed in vertebrate tissues as α1(V)₂α2(V) heterotrimers,¹ which are incorporated into growing fibrils with the more abundant collagen I and involved in regulating the geometry and tensile strength of the resulting collagen I/V heterotypic fibrils.^2,3 Mutations in the genes encoding either the α1(V)⁴ or α2(V)⁵ chain can result in the human heritable connective tissue disorder classic Ehlers-Danlos syndrome (cEDS), clinical hallmarks of which include skin hyperextensibility, atrophic scarring, and joint hypermobility, with patients also often presenting with easy bruising and bleeding.⁶At the molecular level, the collagen fibrils of cEDS skin have variability in diameter not seen in normal skin and include large diameter collagen fibril aggregates with abnormal cauliflower-like shapes when viewed in cross section.⁶ Deficits in the tensile strength of cEDS collagen fibrils are inferred from the hyperextensibility and fragility of cEDS skin and the hypermobility of cEDS joints.Most cEDS cases that have been characterized at the molecular level are heterozygous for null alleles of the α1(V) chain gene COL5A1,⁷ resulting in the deposition of haploinsufficient levels of normal collagen V in tissues, with excess α2(V) chains unable to form stable triple helical molecules or be incorporated into the extracellular matrix (ECM).⁸A lesser number of cEDS cases are associated with COL5A1 missense mutations, and a number of these [eg, signal peptide and C-propeptide mutations that reduce secretion or incorporation of α1(V) chains into heterotrimers, respectively] may result in de facto functional haploinsufficiency rather than structurally abnormal collagen V in the ECM.⁷ An even smaller number of cEDS cases have been associated with missense mutations in the α2(V) chain gene COL5A2 and probably involve incorporation of aberrant collagen 1/V heterotypic fibrils, containing abnormal α2(V) chains, into the ECM.⁷ Interestingly, COL5A2 null alleles have yet to be detected in cEDS patients, leading to the suggestion that haploinsufficiency for the α2(V) chain may not lead to cEDS or, perhaps, to any clinically abnormal phenotype.⁷Previously, knockout of the α1(V) (Col5a1) gene produced a mouse model in which Col5a1^+/− adults exhibit a skin phenotype similar to that of cEDS.^9,10 Col5a1^+/− mice also have decreased aortic stiffness and tensile strength,¹⁰ presumably corresponding to the easy bleeding and somewhat increased prevalence of aortic root dilation, thought to result from increased aortic compliance, in cEDS patients.^11–13 The homozygous null Col5a1^−/− phenotype is embryonic lethal at approximately embryonic day 10, with a seeming absence of collagen fibril formation suggesting an early role for collagen V in a nucleation event necessary to collagen fibril formation.⁹ In another study, mice heterozygous for a small in-frame deletion in the N-telopeptide domain of the α2(V) chain were phenotypically normal, but homozygotes, which developed spinal abnormalities not characteristic of cEDS and most of which died before weaning, had skin with some features reminiscent of cEDS.¹⁴ A subsequent study on the same mice claimed the mutated allele to be functionally null.¹⁵We report the creation and characterization of mice with the first true null Col5a2 allele. Contrary to mice homozygous for the previously described Col5a2 mutant allele,¹⁴ Col5a2^−/− homozygous null mice are early embryonic lethal, consistent with the early embryonic lethality of the previously described Col5a1^−/− mice.⁹ Differences in length of embryonic survival and in collagen fibril density and morphology between the Col5a2^−/− embryos described here and the previously described Col5a1^−/− mice⁹ provide insights into the roles of different forms of collagen V in fibrillogenesis. Col5a2^+/− adults have changes to the extensibility and tensile strength of skin and aortae. Implications of the data for α2(V) and collagen V function and for the possible phenotype of humans heterozygous null for COL5A2 are discussed.     

Novel Roles for Caspase-8 in IL-1β and Inflammasome Regulation

Caspase-8 is an initiator and apical activator caspase that plays a central role in apoptosis. Caspase-8–deficient mice are embryonic lethal, which makes study of caspase-8 in primary immune cells difficult. Recent advances have rescued caspase-8–deficient mice by crossing them to mice deficient in receptor-interacting serine-threonine kinase 3 (RIPK3). These genetic tools have made it possible to study the role of caspase-8 in vivo and in primary immune cells. Several recent studies have identified novel roles for caspase-8 in modulating IL-1β and inflammation, showing that caspase-8 directly regulates IL-1β independent of inflammasomes or indirectly through the regulation of inflammasomes, depending on the stimulus or stimuli that initiate the signaling cascade. Here, we address recent findings on caspase-8 and its role in modulating IL-1β and inflammation.Caspase-8 is an initiator and apical activator caspase that plays a central role in apoptosis. It consists of two N-terminal death effector domains (DEDs), which are followed by a large (p18) and a small (p10) protease subunit at the C-terminal end (Figure 1). First described in 1996, caspase-8 is essential for death receptor–induced activation of the extrinsic cell-death pathway.^1,2 On activation of death receptors (CD95, TNFR1, or DR5), caspase-8 is recruited to the receptors via the adaptor protein FAS-associated death domain (FADD). Caspase-8 and FADD both contain DEDs, which mediate DED–DED homotypic interactions and coordinate complex formation of death receptors. Caspase-8 homodimer formation in this complex results in activation and autocleavage, which further stabilizes the active dimer. Active caspase-8 then processes and cleaves downstream executioner caspases, or the BCL2 family member BID, to initiate apoptosis. Because apoptosis is central for development and survival of the host, caspase-8 activation is tightly regulated. cFLIP, a homolog of caspase-8, blocks caspase-8 apoptotic function by forming heterodimeric complexes³ (Figure 1). It has also been proposed that caspase-8 is cleaved, and in some instances activated by other caspases, such as caspase-3^4,5 and caspase-6,^5,6 as well as by the proteases granzyme B⁷ and cathepsin D.⁸Open in a separate windowFigure 1Role for caspase-8 (CASP8) in inducing apoptosis and regulating signaling pathways. A: Procaspase-8 consists of two N-terminal death-effector domain (DED) prodomains, which are followed by the catalytic subunits p18 and p10, respectively. On dimerization, caspase-8 is cleaved at the sites between the DED and p18, and between p18 and p10. B: Death receptor (CD95, TNFR1, DR5) engagement with the respective ligand [CD95L, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis inducing ligand (TRAIL)] results in recruitment of FAS-associated death domain (FADD) and caspase-8 homodimers. Activation of caspase-8 results in induction of apoptosis. cFLIP can bind to caspase-8 to form cFLIP–caspase-8 heterodimers. The formation of cFLIP–caspase-8 heterodimers inhibits apoptosis. C: Activation of Toll-like receptor 4 (TLR4) or TLR2 results in recruitment of TIR domain-containing adaptor-inducing interferon-β (TRIF) and myeloid differentiation primary response protein MyD88 (MyD88) to the receptors. TRIF and MyD88 signaling results in downstream nuclear factor-κB (NF-κB) signaling events that induce mRNA expression of pro–IL-1β and NLRP3. Evidence suggests that FADD and caspase-8 are required for optimal expression of pro–IL-1β and NLRP3 mRNA, possibly through their role in NF-κB activation. LPS, lipopolysaccharide; PGN, peptidoglycan.The importance of caspase-8 is highlighted by the fact that knockout mice die at approximately embryonic day 10.5.⁹ In seminal studies, the Mocarski¹⁰ and Green¹¹ research groups showed that deletion of receptor-interacting serine-threonine kinase 3 (RIPK3, involved in necroptotic cell death) rescues caspase-8 deficient mice. These studies established a nonapoptotic role for caspase-8, namely, to rescue the lethality induced by RIPK3-mediated pathways. The generation of double-knockout Ripk3^−/−Casp8^−/− mice has provided an invaluable tool for investigating the role of caspase-8 in vivo and in primary immune cells.Here, we discuss inflammasome-mediated IL-1β production and the novel roles of caspase-8 in modulating inflammasomes, IL-1β, and inflammation.     

Peroxisome Proliferator–Activated Receptor α Protects Capillary Pericytes in the Retina

Pericyte degeneration is an early event in diabetic retinopathy and plays an important role in progression of diabetic retinopathy. Clinical studies have shown that fenofibrate, a peroxisome proliferator–activated receptor α (PPARα) agonist, has robust therapeutic effects on diabetic retinopathy in type 2 diabetic patients. We evaluated the protective effect of PPARα against pericyte loss in diabetic retinopathy. In streptozotocin-induced diabetic mice, fenofibrate treatment significantly ameliorated retinal acellular capillary formation and pericyte loss. In contrast, PPARα^−/− mice with diabetes developed more severe retinal acellular capillary formation and pericyte dropout, compared with diabetic wild-type mice. Furthermore, PPARα knockout abolished the protective effect of fenofibrate against diabetes-induced retinal pericyte loss. In cultured primary human retinal capillary pericytes, activation and expression of PPARα both significantly reduced oxidative stress–induced apoptosis, decreased reactive oxygen species production, and down-regulated NAD(P)H oxidase 4 expression through blockade of NF-κB activation. Furthermore, activation and expression of PPARα both attenuated the oxidant-induced suppression of mitochondrial O₂ consumption in human retinal capillary pericytes. Primary retinal pericytes from PPARα^−/− mice displayed more apoptosis, compared with those from wild-type mice under the same oxidative stress. These findings identified a protective effect of PPARα on retinal pericytes, a novel function of endogenous PPARα in the retina.Diabetic retinopathy (DR) is a major sight-threatening microvascular complication of both type 1 and type 2 diabetes.¹ DR is a chronic, progressive, and multifactorial disorder, with retinal microvascular dysfunction being the major component.² Retinal capillary pericytes play an essential role in the maintenance of microvascular stability and regulation of endothelial proliferation.³ It has been shown that pericyte dropout in diabetes correlates with the development of DR.⁴ Pericyte loss is a hallmark of early DR.⁵Relative to healthy subjects, plasma levels of free fatty acids are usually elevated in both type 1 and type 2 diabetic patients.^6,7 Many studies have demonstrated that DR is associated with insulin resistance, which is associated with high plasma free fatty acid levels.^8,9 Palmitate, a saturated fatty acid, has been implicated in dysfunctions and apoptosis in many cell types, including retinal pericytes via activation of NAD(P)H oxidase and NF-κB.^10,11Peroxisome proliferator–activated receptor α (PPARα), a hormone-activated nuclear receptor, is known as an important modulator of lipid metabolism.¹² PPARα has also been shown to have anti-inflammatory and antioxidant activities.^13,14 Fenofibrate, a potent PPARα agonist, has been used clinically to treat dyslipidemia and cardiovascular disease for >30 years.¹⁵ Previous studies support that fenofibrate has anti-inflammatory and antioxidant effects.^16,17 Two recent large, randomized, placebo-controlled clinical trials, The Fenofibrate Intervention in Event Lowering in Diabetes (FIELD) study and The Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, demonstrated robust therapeutic effects of fenofibrate on microvascular complications of diabetes, including DR in type 2 diabetic patients.^18,19 Interestingly, the beneficial effect of fenofibrate on DR was not associated with changes in circulating lipid levels. Our previous study showed that the protective effect of fenofibrate on DR can be achieved through ocular administration, suggesting a local drug target in ocular tissues.²⁰ The mechanism for the protective effect of fenofibrate on DR is not fully elucidated.In our recent studies, we demonstrated that diabetes-induced down-regulation of PPARα plays an important role in retinal inflammation and microvascular dysfunction in DR,²¹ and fenofibrate has therapeutic effects on DR in type 1 diabetes models.²⁰ The function of PPARα in the retina has not been well understood. In this study, we evaluated the protective effect of fenofibrate against pericyte degeneration in DR using primary pericytes, streptozotocin (STZ)-induced diabetic animals, and PPARα knockout (PPARα^−/−) mice, and investigated the underlying molecular mechanism.     

Neutrophil Dependence of Vascular Remodeling after Mycoplasma Infection of Mouse Airways

Vascular remodeling is a feature of sustained inflammation in which capillaries enlarge and acquire the phenotype of venules specialized for plasma leakage and leukocyte recruitment. We sought to determine whether neutrophils are required for vascular remodeling in the respiratory tract by using Mycoplasma pulmonis infection as a model of sustained inflammation in mice. The time course of vascular remodeling coincided with the influx of neutrophils during the first few days after infection and peaked at day 5. Depletion of neutrophils with antibody RB6-8C5 or 1A8 reduced neutrophil influx and vascular remodeling after infection by about 90%. Similarly, vascular remodeling after infection was suppressed in Cxcr2^−/− mice, in which neutrophils adhered to the endothelium of venules but did not extravasate into the tissue. Expression of the venular adhesion molecule P-selectin increased in endothelial cells from day 1 to day 3 after infection, as did expression of the Cxcr2-receptor ligands Cxcl1 and Cxcl2. Tumor necrosis factor α (TNFα) expression increased more than sixfold in the trachea of wild-type and Cxcr2^−/− mice, but intratracheal administration of TNFα did not induce vascular remodeling similar to that seen in infection. We conclude that neutrophil influx is required for remodeling of capillaries into venules in the airways of mice with Mycoplasma infection and that TNFα signaling is necessary but not sufficient for vascular remodeling.Neutrophils are key effector cells of innate immunity that rapidly arrive at sites of tissue injury to kill bacteria and interact with macrophages and other cells to orchestrate a coordinated immune cell and cytokine response to injury.^1–4 Neutrophils are involved in many inflammatory diseases of the airways and lung, including pneumonia, acute lung injury, sepsis, asthma, cystic fibrosis, bronchitis, and chronic obstructive lung disease,⁵ also contribute to tissue damage in inflammatory conditions of other organs, and play a role in arterial remodeling in atherosclerosis.⁴The signals and events that bring neutrophils to sites of inflammation are well characterized.^6–8 These include expression of endothelial cell adhesion molecules to induce rolling and firm attachment, followed by extravasation into tissues where they release cytokines and other products that can kill bacteria and promote tissue remodeling. The dominant mechanism driving neutrophil influx may be organ-specific.^9,10 Blood vessels of the microcirculation undergo numerous changes in sustained inflammation, and these include structural and functional remodeling of endothelial cells and pericytes.^11–14 Among these changes, capillaries transform into venules that support plasma leakage and leukocyte influx. The contribution of neutrophils to this remodeling is not well understood. Circumferential vessel enlargement is a prominent feature of vascular remodeling–sustained airway inflammation^15–23 and is distinct from more familiar and better-documented types of sprouting angiogenesis.²⁴We asked whether incoming neutrophils contribute to the vascular remodeling, with the thought that the initial wave of leukocyte influx could render blood vessels more efficient for leukocyte adhesion and transmigration. Although leukocyte influx is known to accompany blood vessel remodeling,^15,18,22 it is unknown whether there is a causal relationship and, if so, what is the underlying mechanism? Neutrophils are attractive candidates for contributing to vascular remodeling because they are among the first leukocytes to enter inflamed tissues^4,6,25 and can produce cytokines, growth factors, proteases, and reactive oxygen species that have profound vascular effects.^2–4,26With this background, we sought to determine whether neutrophils are essential for the vascular remodeling that occurs soon after Mycoplasma pulmonis infection, when capillaries transform into venules. In particular, we asked whether neutrophil influx coincides spatially and temporally with vascular remodeling, can vascular remodeling be prevented by neutrophil depletion, and if Cxcr2 signaling is required for the neutrophil influx that accompanies vascular remodeling?To address these questions we examined the relationship between neutrophil influx and vascular remodeling during the first week after M. pulmonis infection of the respiratory tract of mice. The approach was to compare the time course of neutrophil influx and vascular remodeling in the trachea and then determine whether the remodeling was blocked by neutrophil depletion by either of two different antineutrophil antibodies: RB6-8C5 or 1A8. We also tested whether vascular remodeling was prevented by genetic deletion of Cxcr2, which mediates the actions of the chemotactic chemokines Cxcl1 and Cxcl2, which bring neutrophils into inflamed tissues. Because previous studies have shown that vascular remodeling was inhibited by blocking tumor necrosis factor α (TNFα) signaling,¹⁹ we asked whether TNFα expression was increased in wild-type and Cxcr2^−/− mice and whether intratracheal administration of TNFα was sufficient to induce vascular remodeling similar to that seen after infection. Other studies examined the expression of the Cxcr2 ligands, Cxcl1 and Cxcl2. Together, the experiments showed that neutrophil influx was required for vascular remodeling after M. pulmonis infection, and that TNF signaling was necessary but not sufficient for vascular remodeling.     

Slam Haplotype 2 Promotes NKT But Suppresses Vγ4+ T-Cell Activation in Coxsackievirus B3 Infection Leading to Increased Liver Damage But Reduced Myocarditis

There are two major haplotypes of signal lymphocytic activation molecule (Slam) in inbred mouse strains, with the Slam haplotype 1 expressed in C57Bl/6 mice and the Slam haplotype 2 expressed in most other commonly used inbred strains, including 129 mice. Because signaling through Slam family receptors can affect innate immunity [natural killer T cell (NKT) and γ-δ T-cell receptor], and innate immunity can determine susceptibility to coxsackievirus B3 (CVB3) infection, the present study evaluated the response of C57Bl/6 and congenic B6.129c1 mice (expressing the 129-derived Slam locus) to CVB3. CVB3-infected C57Bl/6 male mice developed increased myocarditis but reduced hepatic injury compared with infected B6.129c1 mice. C57Bl/6 mice also had increased γδ⁺ and CD8⁺interferon-γ⁺ cells but decreased numbers of NKT (T-cell receptor β chain + mCD1d tetramer⁺) and CD4⁺FoxP3⁺ cells compared with B6.129c1 mice. C57Bl/6 mice were infected with CVB3 and treated with either α-galactosylceramide, an NKT cell-specific ligand, or vehicle (dimethyl sulfoxide/PBS). Mice treated with α-galactosylceramide showed significantly reduced myocarditis. Liver injuries, as determined by alanine aminotransferase levels in plasma, were increased significantly, confirming that NKT cells are protective for myocarditis but pathogenic in the liver.Myocarditis is an inflammation of the cardiac muscle that follows microbial infections.¹ Among viruses, enteroviruses including coxsackie B viruses are common etiologic agents.^2,3 Although infectious agents act as a trigger for myocarditis, there is considerable debate as to the actual mechanism(s) of myocardial injury. Viruses directly cause cellular dysfunction either through induced cell death, shut down of cell RNA and protein synthesis, or viral protease cleavage of contractile proteins.^4,5 In addition, cytokines such as IL-1β, IL-6, and tumor necrosis factor α, which are elicited from resident cells in the heart subsequent to infection, can suppress contractility, leading to cardiac dysfunction.⁶ Finally, host immune responses to infection may kill myocytes, leading to cardiac stress. Host response can be directed specifically toward virally infected cardiocytes or infection can trigger autoimmunity to cardiac antigens (autoimmunity), which destroys both infected and uninfected myocytes.⁷Host innate immune responses occur rapidly, subsequent to viral infections, and usually have broad specificity, unlike the classic adaptive immune response, which requires a week or more for development of a measurable response in the naive individual but is highly specific to the inducing pathogen. The innate immune response both helps to control microbe load before generation of the adaptive immune response and has a major impact on the phenotype and intensity of the adaptive response. Two types of T cells representing innate immunity are natural killer T cells (NKT) and T cells expressing the γ-δ T-cell receptor (γδ⁺). A study by Wu et al⁸ showed that in vivo administration of α-galactosylceramide, a ligand that specifically activates NKT cells, protects mice from coxsackievirus B3 (CVB3)-induced myocarditis. Prior studies have shown that signaling through Slam family receptors has a major impact on NKT cell development,^9–11 and that different Slam haplotypes can have distinct effects on NKT cell response and function.^9,12 There are two major Slam haplotypes, Slam haplotype 1 and Slam haplotype 2, that distinguish commonly used inbred mouse strains.^13,14 Slam haplotype 1 is present in C57Bl/6, and Slam haplotype 2 is present in most other commonly used mouse strains including 129S1/SvImJ and BALB/c mice. The congenic B6.129c1 mouse expresses the genetic region of chromosome 1 containing the 129-derived Slam haplotype 2 locus on the C57Bl/6 background and was used previously to show Slam haplotype control of liver NKT cell numbers and NKT cell cytokine production.¹² In addition, Slam haplotypes previously were shown to regulate macrophage tumor necrosis factor production in response to lipopolysaccharide.¹² Although less well studied, Slam family–receptor signaling also has been shown to affect γδ⁺ T-cell development. Studies using human peripheral blood mononuclear cells stimulated in vitro with antibody to CD3 and either IL-2, anti-CD150 (SLAM), or IL-15 showed that all three stimulation protocols resulted in γδ⁺ T-cell survival. However, co-culture with anti-CD3 and anti-CD150 resulted in selective proliferation of CD8⁺CD56⁺γδ⁺ T cells expressing the Vδ1 chain, and cells co-cultured with anti-CD3 and IL-15 resulted in preferential generation of CD8⁻CD56⁻γδ⁺ cells expressing the Vδ2 chain.¹⁵ Therefore, SLAM signaling can impact the generation of a subpopulation of the total γδ⁺ cell population in humans. Prior studies from the Huber laboratory have shown that a subpopulation of γδ⁺ cells is crucial to myocarditis susceptibility subsequent to CVB3 infection¹⁶ and that the relevant γδ⁺ cell expresses both CD8 and the Vγ4 chain.^16,17 This raised the question of whether Slam haplotypes modulated selected γδ⁺ cell subsets in the mouse, as it does in humans, and whether the Slam haplotype specifically could affect activation of the CD8⁺Vγ4⁺ T cell, which is known to be pathogenic in CVB3-induced myocarditis.CVB3 infection of mice results in multiple organ infection, including pancreas, liver, and heart with accompanying tissue injury in all tissues. There are well-established differences in disease susceptibility between BALB/c and C57Bl/6 mice, strains expressing the two distinct Slam haplotypes. C57Bl/6 mice are highly susceptible to type 2 autoimmune hepatitis and develop extensive hepatic inflammation, whereas BALB/c mice are resistant to this disease and show no inflammation.¹⁸ In contrast, BALB/c mice are more susceptible to myocarditis^19–22 compared with the more resistant C57Bl/6 strain. However, there are many genetic differences between BALB/c and C57Bl/6 mice, which may influence disease development or development and activation of specific innate effectors such as NKT and γδ T cells. The goal of the current study was to determine whether Slam haplotype affected NKT and Vγ4⁺ T-cell responses subsequent to CVB3 infection using C57Bl/6 congenic mice in which the Slam locus alone differed between the mouse strains, and whether haplotype-dependent NKT/Vγ4⁺ cell response had a distinct effect in different organs infected with the virus in the absence of the many other genetic differences between BALB/c and C57Bl/6 mice.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.