A Role for Perforin in Downregulating T-Cell Responses during Chronic Viral Infection

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf −/−) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf −/− mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral “exhaustion” of activated CD8 T cells occurred less efficiently in perf −/− mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf −/− mice; ~50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.     

Role for Complement in the Development of Seizures following Acute Viral Infection

Complement, part of the innate immune system, acts to remove pathogens and unwanted host material. Complement is known to function in all tissues, including the central nervous system (CNS). In this study, we demonstrated the importance of the complement system within the CNS in the development of behavioral seizures following Theiler''s murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice, deficient in complement component C3, developed significantly fewer behavioral seizures following TMEV infection, whereas mice depleted of complement component C3 in the periphery through treatment with cobra venom factor had a seizure rate comparable to that of control mice. These studies indicate that C3 participates in the induction of acute seizures during viral encephalitis.The complement system, a component of the innate immune system, functions to recognize and eliminate pathogens and unwanted host material (1). Activation of complement can occur by the classical, alternative, lectin, and terminal pathways (1). The classical pathway is activated by antigen-antibody complexes, some viruses, Gram-negative bacteria, or C-reactive protein complexes (4). The alternative pathway is activated by lipopolysaccharides and polysaccharides on the surfaces of viruses, bacteria, fungi, and parasites. The lectin pathway is activated by mannose or N-acetylglucosamine on the surfaces of bacteria and other pathogens (4). The complement system, consisting of >40 proteins, is highly regulated by the expression of complement inhibitors and complement receptors, as the complement system can have deleterious effects when unregulated (1). Two important steps in the complement cascade are the cleavage of the multifunctional complement proteins C3 and C5 into C3a and C3b proteins and C5a and C5b proteins, respectively. The anaphylatoxins C3a and C5a function to recruit leukocytes and induce inflammation. C3b functions in opsonization, the process of coating pathogens or particulate material with opsonin and thus making it more susceptible to phagocytosis. C5b functions to initiate the assembly of the C5b-C9 complex, the membrane attack complex (MAC), leading to pathogen lysis. Proteins of the complement system are found throughout all tissues and bodily fluids. Although primarily produced by hepatocytes in the liver, other cell types constitutively express low levels of complement proteins (1).Complement proteins are constitutively produced by neurons, microglia, astrocytes, and oligodendrocytes in the central nervous system (CNS) (14, 16, 44; reviewed in references 1 and 13). Astrocytes, the predominant glial cell type in the brain, are comparable to hepatocytes in terms of the number of complement components they produce (4). The levels of various complement mRNAs and proteins are markedly increased in the CNS following viral infection; for example, C1q and C3 proteins and mRNAs are increased in the rat brain following infection with Borna disease virus (11), and C1q protein and mRNA are increased in the rhesus macaque brain following infection with simian immunodeficiency virus (10). In both cases, the increase in the production of C1q in the brain was localized to microglia/macrophages (10, 11). Complement activation has also been shown to be involved in the control of other viral infections, such as the spread of West Nile virus to the CNS (24).We have been studying the role of the innate immune system in the development of acute behavioral seizures following CNS infection of C57BL/6 mice with a neurotropic virus (19, 23). Infection with Theiler''s murine encephalomyelitis virus (TMEV) results in acute seizures developing in more than 50% of C57BL/6 mice (both male and female) generally between days 3 and 10 postinfection (p.i.) (23). Two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and concomitant inflammatory changes in the brain (perivascular cuffing comprised of infiltrating mononuclear cells, infiltration of macrophages, and/or activation of microglial cells and gliosis) were implicated as contributors to the development of acute seizures (19). In contrast, the proinflammatory cytokine IL-1, TMEV-specific CD8⁺ T cells, and viral persistence were discounted as playing a role in seizures (19). It was found that both the pattern of days on which the mice were observed to have seizures and the seizure score (Racine scale) for any given day varied from mouse to mouse (23). Typically, day 3 p.i. was the first day on which a few mice were observed to have seizures, day 6 p.i. was the peak of behavioral seizure activity, and the majority of seizures had a seizure score of 3 (forelimb clonus) and above (score of 4, rearing; score of 5, rearing and falling). The seizures were afebrile and appeared limbic in nature (23). The seizure frequency was observed to be one per mouse per 2-h observation period, and the duration of the seizures was typically 1 to 2 min (23, 31). Mice experiencing seizures were impaired in both coordination and motor function (23).The role of complement in the development of seizures has been studied in humans with Rasmussen''s encephalitis (RE) (41). Although several different viruses have been detected in the tissues of humans with RE by PCR and in situ hybridization, these results are still controversial. Instead it is thought that an autoimmune process underlies RE. Notwithstanding the unknown etiology of RE, the activation of the complement cascade is thought to be a critical component of disease pathogenesis. Several activated components of the complement system (C4, C8, and MAC) were shown to be present in discrete patches of neurons in the cortex of three out of five patients with active RE by immunohistochemistry (41). As a means of demonstrating in vivo that MAC deposition on neurons could trigger seizures, the individual components of the MAC (C5b6, C7, C8, and C9) were sequentially infused into the rat hippocampus, and assembly of the MAC triggered both behavioral and electrographic seizures as well as cytotoxicity (42).In our current study, we examined the role that complement may play in the development of behavioral seizures in the TMEV-induced seizure model. Through the use of mice deficient in complement component C3 and through depletion of complement component C3 in the periphery, we were able to demonstrate the importance of the complement system within the CNS in the development of seizures in the TMEV-induced seizure model.     

High Viral Fitness during Acute HIV-1 Infection

Several clinical studies have shown that, relative to disease progression, HIV-1 isolates that are less fit are also less pathogenic. The aim of the present study was to investigate the relationship between viral fitness and control of viral load (VL) in acute and early HIV-1 infection. Samples were obtained from subjects participating in two clinical studies. In the PULSE study, antiretroviral therapy (ART) was initiated before, or no later than six months following seroconversion. Subjects then underwent multiple structured treatment interruptions (STIs). The PHAEDRA study enrolled and monitored a cohort of individuals with documented evidence of primary infection. The subset chosen were individuals identified no later than 12 months following seroconversion to HIV-1, who were not receiving ART. The relative fitness of primary isolates obtained from study participants was investigated ex vivo. Viral DNA production was quantified using a novel real time PCR assay. Following intermittent ART, the fitness of isolates obtained from 5 of 6 PULSE subjects decreased over time. In contrast, in the absence of ART the fitness of paired isolates obtained from 7 of 9 PHAEDRA subjects increased over time. However, viral fitness did not correlate with plasma VL. Most unexpected was the high relative fitness of isolates obtained at Baseline from PULSE subjects, before initiating ART. It is widely thought that the fitness of strains present during the acute phase is low relative to strains present during chronic HIV-1 infection, due to the bottleneck imposed upon transmission. The results of this study provide evidence that the relative fitness of strains present during acute HIV-1 infection may be higher than previously thought. Furthermore, that viral fitness may represent an important clinical parameter to be considered when deciding whether to initiate ART during early HIV-1 infection.     

MicroRNA-324-3p Plays A Protective Role Against Coxsackievirus B3-Induced Viral Myocarditis

Virologica Sinica - Viral myocarditis (VM) is an inflammatory disease of the myocardium associated with heart failure, which is caused by common viral infections. A majority of the infections are...     

Astrocyte-Derived CXCL10 Drives Accumulation of Antibody-Secreting Cells in the Central Nervous System during Viral Encephalomyelitis

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10^−/− but not CXCL9^−/− mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10^−/− mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9^−/− mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9^−/− or CXCL10^−/− mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.     

苦参碱注射液对急性病毒性心肌炎的治疗作用

目的：观察苦参碱注射液治疗急性病毒性心肌炎（AVM）的临床疗效。方法：将AVM患者120例随机分为治疗组63例及对照组57例,对照组采用常规疗法,治疗组在对照组基础上给予苦参碱注射液治疗,连用2周。观察临床疗效。结果：治疗组总有效率90.5%,对照组总有效率73.7%,两组比较有显著性差异。治疗组治疗后血清肌酸磷酸激酶（CPK）、天冬氨酸转氨酶（AST）及血清乳酸脱氢酶（LDH）较对照组治疗后明显降低,差异有统计学意义（P〈0.05）。结论：苦参碱对急性心肌炎有良好疗效。     

苦参碱注射液对急性病毒性心肌炎的治疗作用

目的:观察苦参碱注射液治疗急性病毒性心肌炎(AVM)的临床疗效.方法:将AVM患者120例随机分为治疗组63例及对照组57例,对照组采用常规疗法,治疗组在对照组基础上给予苦参碱注射液治疗,连用2周.观察临床疗效.结果:治疗组总有效率90.5%,对照组总有效率73.7%,两组比较有显著性差异.治疗组治疗后血清肌酸磷酸激酶(CPK)、天冬氨酸转氨酶(AST)及血清乳酸脱氢酶(LDH)较对照组治疗后明显降低,差异有统计学意义(P＜0.05).结论:苦参碱对急性心肌炎有良好疗效.     

Protective Role of the Cholinergic Anti-Inflammatory Pathway in a Mouse Model of Viral Myocarditis

Background

Activation of the cholinergic anti-inflammatory pathway, which relies on the α7nAchR (alpha 7 nicotinic acetylcholine receptor), has been shown to decrease proinflammatory cytokines. This relieves inflammatory responses and improves the prognosis of patients with experimental sepsis, endotoxemia, ischemia/reperfusion injury, hemorrhagic shock, pancreatitis, arthritis and other inflammatory syndromes. However, whether the cholinergic anti-inflammatory pathway has an effect on acute viral myocarditis has not been investigated. Here, we studied the effects of the cholinergic anti-inflammatory pathway on acute viral myocarditis.

Methodology/Principal Findings

In a coxsackievirus B3 murine myocarditis model (Balb/c), nicotine and methyllycaconitine were used to stimulate and block the cholinergic anti-inflammatory pathway, respectively. Relevant signal pathways were studied to compare their effects on myocarditis, survival rate, histopathological changes, ultrastructural changes, and cytokine levels. Nicotine treatments significantly improved survival rate, attenuated myocardial lesions, and downregulated the expression of TNF-α and IL-6. Methyllycaconitine decreased survival rate, aggravated myocardial lesions, and upregulated the expression of TNF-α and IL-6. In addition, levels of the signaling protein phosphorylated STAT3 were higher in the nicotine group and lower in the methyllycaconitine group compared with the untreated myocarditis group.

Conclusions/Significance

These results show that nicotine protects mice from CVB3-induced viral myocarditis and that methyllycaconitine aggravates viral myocarditis in mice. Because nicotine is a α7nAchR agonist and methyllycaconitine is a α7nAchR antagonist, we conclude that α7nAchR activation increases the phosphorylation of STAT3, reduces the expression of TNF-α and IL-6, and, ultimately, alleviates viral myocarditis. We also conclude that blocking α7nAchR reduces the phosphorylation of STAT3, increases the expression of TNF-α and IL-6, aggravating viral myocarditis.     

A Novel Host-Proteome Signature for Distinguishing between Acute Bacterial and Viral Infections

Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

Essential Role of NK Cells in IgG Therapy for Experimental Autoimmune Encephalomyelitis

Intravenous immunoglobulin has long been used in treating autoimmune diseases, although mechanisms remain uncertain. Activating Fcγ receptors are receptors of IgG and reported to be essential in intravenous immunoglobulin (IVIG) therapy. Therefore, we hypothesized natural killer (NK) cells, which express abundant activating Fcγ receptors, are the potential cellular target. In experimental autoimmune encephalomyelitis (EAE), we demonstrated that IgG suppressed disease development in intact, but not in NK cell depleted mice. Adoptive transfer of IgG-treated NK cell could protect mice against EAE, and suppressed interferon γ and interleukin 17 production. The percentage of CD4⁺Foxp3⁺ regulatory T cells was significantly increased. The increase of regulatory T cells was also observed in IgG-treated EAE mice but not in NK cell depleted mice. In vitro experiments confirmed that IgG-treated NK cells enhanced regulatory T cell induction from naïve CD4⁺ T cells. Interestingly, cells from draining lymph nodes produced more interleukin 2 after the adoptive transfer of IgG-treated NK cells. We neutralized interleukin 2 and the induction of CD4⁺Foxp3⁺ T cells by IgG-treated NK cells was significantly reduced. To our knowledge, we identified for the first time the critical role of NK cells in the mechanism of IgG-induced induction of Treg cells in treatment of autoimmunity.     

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.     

A Non-Neuronal Cardiac Cholinergic System Plays a Protective Role in Myocardium Salvage during Ischemic Insults

Background

In our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system–cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system–it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated.

Aim

We investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes.

Results

Choline acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell–cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3.

Conclusion

The non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.     

HIV-1 Vpr Accelerates Viral Replication during Acute Infection by Exploitation of Proliferating CD4+ T Cells In Vivo

The precise role of viral protein R (Vpr), an HIV-1-encoded protein, during HIV-1 infection and its contribution to the development of AIDS remain unclear. Previous reports have shown that Vpr has the ability to cause G₂ cell cycle arrest and apoptosis in HIV-1-infected cells in vitro. In addition, vpr is highly conserved in transmitted/founder HIV-1s and in all primate lentiviruses, which are evolutionarily related to HIV-1. Although these findings suggest an important role of Vpr in HIV-1 pathogenesis, its direct evidence in vivo has not been shown. Here, by using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrated that Vpr causes G₂ cell cycle arrest and apoptosis predominantly in proliferating CCR5⁺ CD4⁺ T cells, which mainly consist of regulatory CD4⁺ T cells (Tregs), resulting in Treg depletion and enhanced virus production during acute infection. The Vpr-dependent enhancement of virus replication and Treg depletion is observed in CCR5-tropic but not CXCR4-tropic HIV-1-infected mice, suggesting that these effects are dependent on the coreceptor usage by HIV-1. Immune activation was observed in CCR5-tropic wild-type but not in vpr-deficient HIV-1-infected humanized mice. When humanized mice were treated with denileukin diftitox (DD), to deplete Tregs, DD-treated humanized mice showed massive activation/proliferation of memory T cells compared to the untreated group. This activation/proliferation enhanced CCR5 expression in memory CD4⁺ T cells and rendered them more susceptible to CCR5-tropic wild-type HIV-1 infection than to vpr-deficient virus. Taken together, these results suggest that Vpr takes advantage of proliferating CCR5⁺ CD4⁺ T cells for enhancing viremia of CCR5-tropic HIV-1. Because Tregs exist in a higher cycling state than other T cell subsets, Tregs appear to be more vulnerable to exploitation by Vpr during acute HIV-1 infection.