Inflammasome activation occurs in CD4+ and CD8+ T cells during graft-versus-host disease.

A severe complication of hematopoietic stem cell transplantation is graft-versus-host disease (GvHD), a reaction that occurs following the transfer of donor immune cells (the graft) into an allogeneic host. Transplanted cells recognize host alloantigens as foreign, resulting in the activation of donor T cells and migration of these pathological cells into host tissues. In this study, we found that caspase-1 is activated in alloreactive murine and human CD4+ and CD8+ T cells early during acute GvHD (aGvHD). The presence of inflammasome-bound active caspase-1 (p33) and ASC-speck formation confirmed inflammasome activation in these cells. We further measured gasdermin D (GSDMD) cleavage and IL-18 secretion from alloreactive T cells ex vivo. Isolated T cells with high levels of active caspase-1 had a strong inflammatory transcriptional signature and a metabolic phenotype similar to inflammatory myeloid cells, including the upregulation of proinflammatory cytokines and metabolic switch from oxidative phosphorylation to aerobic glycolysis. We also observed oxidative stress, mitochondrial dysfunction, and cell death phenotypes consistent with inflammatory cell death in alloreactive T cells. For the first time, this study characterizes caspase-1 activation in transplanted T cells during aGvHD, using mouse and human models, adding to a body of literature supporting inflammasome function in cells of the adaptive immune system.

Repeated closed-head mild traumatic brain injury-induced inflammation is associated with nociceptive sensitization.

BACKGROUND: Individuals who have experienced mild traumatic brain injuries (mTBIs) suffer from several comorbidities, including chronic pain. Despite extensive studies investigating the underlying mechanisms of mTBI-associated chronic pain, the role of inflammation in long-term pain after mTBIs is not fully elucidated. Given the shifting dynamics of inflammation, it is important to understand the spatial-longitudinal changes in inflammatory processes following mTBIs and their effects on TBI-related pain. METHODS: We utilized a recently developed transgenic caspase-1 luciferase reporter mouse model to monitor caspase-1 activation through a thinned skull window in the in vivo setting following three closed-head mTBI events. Organotypic coronal brain slice cultures and acutely dissociated dorsal root ganglion (DRG) cells provided tissue-relevant context of inflammation signal. Mechanical allodynia was assessed by mechanical withdrawal threshold to von Frey and thermal hyperalgesia withdrawal latency to radiant heat. Mouse grimace scale (MGS) was used to detect spontaneous or non-evoked pain. In some experiments, mice were prophylactically treated with MCC950, a potent small molecule inhibitor of NLRP3 inflammasome assembly to inhibit injury-induced inflammatory signaling. Bioluminescence spatiotemporal dynamics were quantified in the head and hind paws, and caspase-1 activation was confirmed by immunoblot. Immunofluorescence staining was used to monitor the progression of astrogliosis and microglial activation in ex vivo brain tissue following repetitive closed-head mTBIs. RESULTS: Mice with repetitive closed-head mTBIs exhibited significant increases of the bioluminescence signals within the brain and paws in vivo for at least one week after each injury. Consistently, immunoblotting and immunofluorescence experiments confirmed that mTBIs led to caspase-1 activation, astrogliosis, and microgliosis. Persistent changes in MGS and hind paw withdrawal thresholds, indicative of pain states, were observed post-injury in the same mTBI animals in vivo. We also observed enhanced inflammatory responses in ex vivo brain slice preparations and DRG for at least 3 days following mTBIs. In vivo treatment with MCC950 significantly reduced caspase-1 activation-associated bioluminescent signals in vivo and decreased stimulus-evoked and non-stimulus evoked nociception. CONCLUSIONS: Our findings suggest that the inflammatory states in the brain and peripheral nervous system following repeated mTBIs are coincidental with the development of nociceptive sensitization, and that these events can be significantly reduced by inhibition of NLRP3 inflammasome activation.

Monitoring of inflammation using novel biosensor mouse model reveals tissue- and sex-specific responses to Western diet.

Obesity is an epidemic, and it is characterized by a state of low-grade systemic inflammation. A key component of inflammation is the activation of inflammasomes, multiprotein complexes that form in response to danger signals and that lead to activation of caspase-1. Previous studies have found that a Westernized diet induces activation of inflammasomes and production of inflammatory cytokines. Gut microbiota metabolites, including the short-chain fatty acid butyrate, have received increased attention as underlying some obesogenic features, but the mechanisms of action by which butyrate influences inflammation in obesity remain unclear. We engineered a caspase-1 reporter mouse model to measure spatiotemporal dynamics of inflammation in obese mice. Concurrent with increased capsase-1 activation in vivo, we detected stronger biosensor signal in white adipose and heart tissues of obese mice ex vivo and observed that a short-term butyrate treatment affected some, but not all, of the inflammatory responses induced by Western diet. Through characterization of inflammatory responses and computational analyses, we identified tissue- and sex-specific caspase-1 activation patterns and inflammatory phenotypes in obese mice, offering new mechanistic insights underlying the dynamics of inflammation.

DSS-induced inflammation in the colon drives a proinflammatory signature in the brain that is ameliorated by prophylactic treatment with the S100A9 inhibitor paquinimod.

BACKGROUND: Inflammatory bowel disease (IBD) is established to drive pathological sequelae in organ systems outside the intestine, including the central nervous system (CNS). Many patients exhibit cognitive deficits, particularly during disease flare. The connection between colonic inflammation and neuroinflammation remains unclear and characterization of the neuroinflammatory phenotype in the brain during colitis is ill-defined. METHODS: Transgenic mice expressing a bioluminescent reporter of active caspase-1 were treated with 2% dextran sodium sulfate (DSS) for 7 days to induce acute colitis, and colonic, systemic and neuroinflammation were assessed. In some experiments, mice were prophylactically treated with paquinimod (ABR-215757) to inhibit S100A9 inflammatory signaling. As a positive control for peripheral-induced neuroinflammation, mice were injected with lipopolysaccharide (LPS). Colonic, systemic and brain inflammatory cytokines and chemokines were measured by cytokine bead array (CBA) and Proteome profiler mouse cytokine array. Bioluminescence was quantified in the brain and caspase activation was confirmed by immunoblot. Immune cell infiltration into the CNS was measured by flow cytometry, while light sheet microscopy was used to monitor changes in resident microglia localization in intact brains during DSS or LPS-induced neuroinflammation. RNA sequencing was performed to identify transcriptomic changes occurring in the CNS of DSS-treated mice. Expression of inflammatory biomarkers were quantified in the brain and serum by qRT-PCR, ELISA and WB. RESULTS: DSS-treated mice exhibited clinical hallmarks of colitis, including weight loss, colonic shortening and inflammation in the colon. We also detected a significant increase in inflammatory cytokines in the serum and brain, as well as caspase and microglia activation in the brain of mice with ongoing colitis. RNA sequencing of brains isolated from DSS-treated mice revealed differential expression of genes involved in the regulation of inflammatory responses. This inflammatory phenotype was similar to the signature detected in LPS-treated mice, albeit less robust and transient, as inflammatory gene expression returned to baseline following cessation of DSS. Pharmacological inhibition of S100A9, one of the transcripts identified by RNA sequencing, attenuated colitis severity and systemic and neuroinflammation. CONCLUSIONS: Our findings suggest that local inflammation in the colon drives systemic inflammation and neuroinflammation, and this can be ameliorated by inhibition of the S100 alarmin, S100A9.

Amelioration of Graft-versus-Host Disease by Exopolysaccharide from a Commensal Bacterium.

Acute graft-versus-host disease (aGvHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pretransplantation therapy, up to 60% of these patients develop aGvHD, and require additional immunosuppressive intervention. We treated mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, shortly before and after induction of aGvHD and found that, whereas only 10% of control mice survived to day 80, 70% of EPS-treated mice survived to 80 d. EPS treatment of donor-only mice resulted in ∼60% survival. Using a biosensor mouse model to assess inflammation in live mice during aGvHD, we found that EPS prevented the activation of alloreactive donor T cells. In vitro, EPS did not affect T cells directly but, instead, induced bone marrow-derived dendritic cells (BMDCs) that displayed characteristics of inhibitory dendritic cells (DCs). Development of these BMDCs required TLR4 signaling through both MyD88 and TRIF pathways. Using BMDCs derived from IDO knockout mice, we showed that T cell inhibition by EPS-treated BMDCs was mediated through the suppressive effects of IDO. These studies describe a bacterial molecule that modulates immune responses by inducing inhibitory DCs in a TLR4-dependent manner, and these cells have the capacity to inhibit T cell activation through IDO. We suggest that EPS or EPS-treated DCs can serve as novel agents for preventing aGvHD.

Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus.

Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein1 to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex2-4. Although recent studies have observed the presence of the capsid protein in the nucleus during infection5-8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.

A Caspase-1 Biosensor to Monitor the Progression of Inflammation In Vivo.

Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro-IL-1ß and pro-IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow-derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.

K63-Linked Ubiquitin Is Required for Restriction of HIV-1 Reverse Transcription and Capsid Destabilization by Rhesus TRIM5α.

TRIM5α is an antiviral restriction factor that inhibits retroviral infection in a species-specific fashion. TRIM5α binds to and forms assemblies around the retroviral capsid. Following binding, poorly understood, ubiquitin-dependent events lead to the disassembly of the viral core, prior to the accumulation of viral reverse transcription products in the target cell. It is also known that assemblies of TRIM5α and other TRIM family proteins can be targets of autophagic degradation. The goal of this study was to define the role of specific ubiquitin linkages in the retroviral restriction and autophagic degradation of TRIM5α and delineate any connection between these two processes. To this end, we generated fusion proteins in which the catalytic domains of different deubiquitinase (DUB) enzymes, with different specificities for polyubiquitinated linkages, were fused to the N-terminal RING domain of Rhesus macaque TRIM5α. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5α with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5α is required to induce capsid disassembly and to inhibit reverse transcription of HIV, while the ability to inhibit HIV-1 infection was not dependent on K63-linked ubiquitination. We also observed that K63-linked ubiquitination is required for the association of TRIM5α with autophagosomal membranes and the autophagic adapter protein p62.IMPORTANCE Although the mechanisms by which TRIM5α can induce the abortive disassembly of retroviral capsids have remained obscure, numerous studies have suggested a role for ubiquitination and cellular degradative pathways. These studies have typically relied on global perturbation of cellular degradative pathways. Here, through the use of linkage-specific deubiquitinating enzymes tethered to TRIM5α, we delineate the ubiquitin linkages which drive specific steps in restriction and degradation by TRIM5α, providing evidence for a noncanonical role for K63-linked ubiquitin in the process of retroviral restriction by TRIM5α and potentially providing insight into the mechanism of action of other TRIM family proteins.

KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection.

Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection.

TRIM5α Degradation via Autophagy Is Not Required for Retroviral Restriction.

UNLABELLED: TRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins. IMPORTANCE: Restriction factors are a class of proteins that inhibit viral replication. Following fusion of a retrovirus with a host cell membrane, the retroviral capsid is released into the cytoplasm of the target cell. TRIM5α inhibits retroviral infection by promoting the abortive disassembly of incoming retroviral capsid cores; as a result, the retroviral genome is unable to traffic to the nucleus, and the viral life cycle is extinguished. In the process of restriction, TRIM5α itself is degraded by the proteasome. However, in the present study, we have shown that in the absence of a restriction-sensitive virus, TRIM5α is degraded by both proteasomal and autophagic degradation pathways. Notably, we observed that restriction of retroviruses by TRIM5α does not require autophagic machinery. These data indicate that the effector functions of TRIM5α can be separated from its degradation and may have further implications for understanding the mechanisms of other TRIM family members.

TRIM5α-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization.

UNLABELLED: Rhesus macaque TRIM5α (rhTRIM5α) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5α restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5α, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5α fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5α. Similarly, expression of DUb-rhTRIM5α resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5α induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5α-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5α failed to activate NF-κB signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection. IMPORTANCE: These studies provide direct evidence that ubiquitin conjugation to rhTRIM5α-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action.

Genome Sequences of Six Paenibacillus larvae Siphoviridae Phages.

Six sequenced and annotated genomes of Paenibacillus larvae phages isolated from the combs of American foulbrood-diseased beehives are 37 to 45 kbp and have approximately 42% G+C content and 60 to 74 protein-coding genes. Phage Lily is most divergent from Diva, Rani, Redbud, Shelly, and Sitara.