PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical ß-coronavirus disease.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.

In Vivo Photodynamic Therapy With a Lipophilic Zinc(II) Phthalocyanine Inhibits Colorectal Cancer and Induces a Th1/CD8 Antitumor Immune Response.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is an antitumor procedure clinically approved for the treatment of different cancer types. Despite strong efforts and promising results in this field, PDT has not yet been approved by any regulatory authority for the treatment of colorectal cancer, one of the most prevalent gastrointestinal tumors. In the search of novel therapeutic strategies, we examined the in vivo effect of PDT with a lipophilic phthalocyanine (Pc9) encapsulated into polymeric poloxamine micelles (T1107) in a murine colon carcinoma model. STUDY DESIGN/MATERIALS AND METHODS: In vivo assays were performed with BALB/c mice challenged with CT26 cells. Pc9 tumor uptake was evaluated with an in vivo imaging system. Immunofluorescence, western blot, and flow cytometry assays were carried out to characterize the activation of apoptosis and an antitumor immune response. RESULTS: Pc9-T1107 effectively delayed tumor growth and prolonged mice survival, without generating systemic or tissue-specific toxicity. The induction of an apoptotic response was characterized by a decrease in the expression levels of Bcl-XL , Bcl-2, procaspase 3, full length Bid, a significant increment in the amount of active caspase-3 and the detection of PARP-1 cleavage. Infiltration of CD8+ CD107a+ T cells and higher levels of interferon-γ and tumor necrosis factor-α were also found in PDT-treated tumors. CONCLUSIONS: Pc9-T1107 PDT treatment reduced tumor growth, inducing an apoptotic cell death and activating an immune response. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

The autoimmune response elicited by mouse hepatitis virus (MHV-A59) infection is modulated by liver tryptophan-2,3-dioxygenase (TDO).

In a previous work we demonstrated that inhibition of mouse indoleamine 2,3-dioxygenase (IDO) by methyltryptophan (MT) exacerbated the pathological actions of mouse hepatitis virus (MHV-A59) infection, suggesting that tryptophan (TRP) catabolism was involved in viral effects. Since there is a second enzyme that dioxygenates TRP, tryptophan-2, 3-dioxygenase (TDO), which is mainly located in liver, we decided to study its role in our model of MHV-infection. Results showed that in vivo TDO inhibition by LM10, a derivative of 3-(2-(pyridyl) ethenyl) indole, resulted in a decrease of anti- MHV Ab titers induced by the virus infection. Besides, a reduction of some alarmin release, i.e, uric acid and high-mobility group box1 protein (HMGB1), was observed. Accordingly, since alarmin liberation was related to the expression of autoantibodies (autoAb) to fumarylacetoacetate hydrolase (FAH), these autoAb also diminished. Moreover, PCR results indicated that TDO inhibition did not abolish viral replication. Furthermore, histological liver examination did not reveal strong pathologies, whereas mouse survival was hundred percent in control as well as in MHV-infected mice treated with LM10. Data presented in this work indicate that in spite of the various TDO actions already described, specific TDO blockage could also restrain some MHV actions, mainly suppressing autoimmune reactions. Such results should prompt further experiments with various viruses to confirm the possible use of a TDO inhibitor such as LM-10 to treat either viral infections or even autoimmune diseases triggered by a viral infection.

Environmental Cues Modulate Microglial Cell Behavior Upon Shiga Toxin 2 From Enterohemorrhagic Escherichia coli Exposure.

Shiga toxin (Stx) produced by enterohemorrhagic E. coli produces hemolytic uremic syndrome and encephalopathies in patients, which can lead to either reversible or permanent neurological abnormalities, or even fatal cases depending on the degree of intoxication. It has been observed that the inflammatory component plays a decisive role in the severity of the disease. Therefore, the objective of this work was to evaluate the behavior of microglial cell primary cultures upon Stx2 exposure and heat shock or lipopolysaccharide challenges, as cues which modulate cellular environments, mimicking fever and inflammation states, respectively. In these contexts, activated microglial cells incorporated Stx2, increased their metabolism, phagocytic capacity, and pro-inflammatory profile. Stx2 uptake was associated to receptor globotriaosylceramide (Gb3)-pathway. Gb3 had three clearly distinguishable distribution patterns which varied according to different contexts. In addition, toxin uptake exhibited both a Gb3-dependent and a Gb3-independent binding depending on those contexts. Altogether, these results suggest a fundamental role for microglial cells in pro-inflammatory processes in encephalopathies due to Stx2 intoxication and highlight the impact of environmental cues.

Effects of interleukin 17A (IL-17A) neutralization on murine hepatitis virus (MHV-A59) infection.

Mice infected with mouse hepatitis virus A59 (MHV-A59) develop hepatitis and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH), a fact closely related to the release of alarmins such as uric acid and/or high-mobility group box protein 1 (HMGB1). We studied the effect of neutralizing monoclonal antibodies (MAb) against IL-17A in our model of mouse MHV-A59-infection. MAb anti-IL-17F and anti-IFNγ were used to complement the study. Results showed that transaminase levels markedly decreased in MHV-A59-infected mice treated with MAb anti-IL-17A whereas plasmatic Ig concentration sharply increased. Conversely, MAb anti-IL-17F enhanced transaminase liberation and did not affect Ig levels. Serum IFNγ was detected in mice infected with MHV-A59 and its concentration increased after MAb anti-IL-17A administration. Besides, MAb anti-IFNγ greatly augmented transaminase plasmatic levels. IL-17A neutralization did not affect MHV-A59-induction of HMGB1 liberation and slightly augmented plasmatic uric acid concentration. However, mice treated with the MAb failed to produce autoAb to FAH. The above results suggest a reciprocal regulation of Th1 and Th17 cells acting on the different MHV-A59 effects. In addition, it is proposed that IL-17A is involved in alarmins adjuvant effects leading to autoAb expression.

Levo-1-methyl tryptophan aggravates the effects of mouse hepatitis virus (MHV-A59) infection.

Mice infected with mouse hepatitis virus A59 (MHV-A59) develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH) with a concomitant enhancement of transaminases and release of alarmins such as uric acid and high-mobility group box protein 1 (HMGB1). Tryptophan catabolism is an endogenous mechanism that restricts excessive immune responses, thereby preventing immunopathology. Since indoleamine-2,3-dioxygenase (IDO) is the key and rate-limiting enzyme of tryptophan catabolism, the aim of this work was to explore whether specific inhibition of IDO by Levo-1-methyl tryptophan (MT) could affect MHV actions. Results showed that MT strongly enhanced the hypergammaglobulinemia induced by the virus, as well as anti-MHV Ab and uric acid release. Moreover, infected mice treated with MT did express anti-FAH autoAb and high levels of serum HMGB1. Survival of MHV-infected animals treated with MT was severely reduced compared with that of MHV-infected mice or controls only treated with MT. Furthermore, histological liver examination indicated that MT induced fibrosis in MHV-infected animals, whereas MT itself increased uric acid levels without shortening the animal life Thus, under our experimental conditions, results indicated an exacerbated response to MHV infection when IDO was blocked by MT.

Uric acid and HMGB1 are involved in the induction of autoantibodies elicited in mice infected with mouse hepatitis virus A59.

We have shown that mice infected with mouse hepatitis virus A59 develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH). Because it has been proposed that the immune system is stimulated by alarm signals called damage-associated molecular patterns or alarmins, we investigated the participation of uric acid and high-mobility group box protein 1 (HMGB1) in the autoimmune response elicited by mouse hepatitis virus (MHV). Mice subjected to MHV infection had increased plasmatic uric acid concentration that significantly decreased after 20 days of daily treatment with allopurinol and, simultaneously, autoAb to FAH were undetected. Furthermore, this autoAb disappeared after 30 days of treatment with ethyl pyruvate, along with a substantial reduction in serum HMGB1 concentration. Both results indicated a remarkable relationship between the autoimmune process induced by the virus and uric acid and HMGB1 liberation. Unexpectedly, it was found that allopurinol and ethyl pyruvate inhibited the release of both uric acid and HMGB1. Because HMGB1 is activated through binding to interleukin 1ß, and that this cytokine is produced by the NLRP3 inflammasome that could be stimulated by uric acid, we propose that both alarmins could be acting in concert with the induction of the autoAb to FAH in MHV-infected mice.

Fine specificity of autoantibodies induced by mouse hepatitis virus A59.

We have shown that mice infected with mouse hepatitis virus A59 (MHV-A59) develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH). The autoAb recognized conformational as well as linear antigenic determinants in the enzyme, and the autoimmune response was not entirely restricted to molecular mimicry and/or epitope spreading. Since the N- and C-terminal portions of the enzyme were the most reactive with autoAb, the fine specificity of these Ab was investigated. Immobilized 15-mer linear peptides (overlapping by 14 amino acids) spanning the N-terminal FAH sequence 1-49 were recognized by Ab from MHV-infected mice. The pattern of reactivity indicated the existence of two major epitope cores (i.e., sequences 9-23 and 30-44), and sequence comparison permitted the identification of two minimal epitopes, DSDFPIQ (amino acids 9-15) and IGDQILD (amino acids 36-42). Mutational analysis of sequences 9-23 and 30-44 indicated that residues 9-12 (DSDF) from the first major N-terminal epitope, and residue 36 (I) from the second, were the key amino acids energetically important for Ab contact. Interestingly, those residues were inside the two minimal epitopes previously predicted. The C-terminal portion of the enzyme (sequence 390-419) presented only one major epitope, located between residues 390 and 409. In this case, the minimal epitope had nine amino acids, CQGDGYRVG, corresponding to the FAH sequence 396-404 that outlines a loop specific for the enzyme. Data indicated that neither the FAH minimal epitopes nor the key residues important for binding to Ab from MHV-infected mice have their counterparts in the viral proteins. However, location of the energetically important residues in the tertiary structure of the enzyme originates a virtual conformational epitope. Such hypothetical B-cell epitopes could be present in any viral protein, originating a cross-reaction leading to the autoimmune response induced by MHV.

The autoimmune response induced by mouse hepatitis virus A59 is expanded by a hepatotoxic agent.

Mouse hepatitis virus strain A59 (MHV-A59) triggers various pathologies in several mouse strains, including hypergammaglobulinaemia, hepatitis and thymus involution. We reported previously the presence of autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH) in sera from mice infected with MHV-A59. Long-term MHV-infected mice represented a good model of non-pathogenic autoimmune response since the animals were apparently healthy in spite of the presence of autoAb. The aim of this work was to see whether a severe liver injury, which releases endogenous adjuvants, i.e. danger signals, could elicit a broader spectrum of autoAb and perhaps signs of autoimmune hepatitis. Carbon tetrachloride (CCl(4)) was injected into mice 30 days after MHV infection, and serum was assayed for autoAb and total IgG 20 days later. The association of MHV infection with the toxic effects of CCl(4) resulted in hypergammaglobulinaemia and the production of autoAb to various liver and kidney proteins. Histological examination of liver samples showed tissue damages but without significant differences between the animals submitted to MHV+CCl(4) and controls, which were either infected by MHV without CCl(4), or poisoned by CCl(4) in the absence of MHV infection. Those results show that liver injury after viral infection may lead to the spreading of the immune response and to an increase of serum IgG, suggesting that the procedure used herein could simulate the onset of autoimmune hepatitis.

The peptide specificities of the autoantibodies elicited by mouse hepatitis virus A59.

Synthetic decapeptides (N=206) covering the entire sequence of mouse liver fumarylacetoacetate hydrolase (FAH) were used to analyze the specificities of the autoantibodies (autoAb) elicited towards this enzyme in mice infected with mouse hepatitis virus (MHV). These autoAb bound mainly to N- and C-terminal FAH peptides, the most reactive sequences being 1-50 and 390-420, respectively. Surprisingly, although FAH sequence 1-50 shares a high degree of homology with various MHV proteins, the C-terminal portion does not. Moreover, whereas the autoAb reacted with homologous peptides surrounding residues 70, 160 and 360, non-similar sequences around residues 130, 210, 240, 250, and 300 were also recognized, indicating that autoAb were not restricted to epitopes with sequence homologies. There was also a lack of correlation between the amount of anti-MHV or anti-FAH antibodies produced and the reactivity towards the peptides. Moreover, the spectrum of peptides recognized by the autoAb of a given mouse did not change significantly with time, which suggests that the MHV-elicited autoimmune response does not induce an epitope recognition spreading. Finally, anti-FAH Ab produced after immunization with rat liver FAH recognized essentially the same mouse FAH regions than autoAb from MHV-infected mice. Results indicated that the induction of the autoAb is not only related to molecular or structural mimicry, but rather supports the Danger model, in which any aggression, in this case the MHV infection, is susceptible to trigger the production of autoAb.

A low chronic ethanol exposure induces morphological changes in the adolescent rat brain that are not fully recovered even after a long abstinence: an immunohistochemical study.

Little is known about the morphological effects of alcoholism on the developing adolescent brain and its consequences into adulthood. We studied here the relationship between two neurotransmitter systems (the serotoninergic and nitrergic) and the astrocytic and neuronal cytoskeleton immediately and long after drinking cessation of a chronic, but low, ethanol administration. Adolescent male Wistar rats were exposed to ethanol 6.6% (v/v) in drinking water for 6 weeks and studied after ending exposure or after a 10-week recovery period drinking water. Control animals received water. Brain sections were processed by immunohistochemistry using antibodies to serotonin (5-HT); glial fibrillary acidic protein (GFAP); astroglial S-100b protein; microtubule associated protein-2 (MAP-2); 200 kDa neurofilaments (Nf-200); and neuronal nitric oxide synthase (nNOS). The mesencephalic dorsal and median raphe nucleus (DRN; MRN) and three prosencephalic areas closely related to cognitive abilities (CA1 hippocampal area, striatum and frontal cortex) were studied by digital image analysis. 5-HT immunoreactivity (-ir) decreased in the DRN and recovered after abstinence and was not changed in the MRN. In the three prosencephalic areas, astrocytes' cell area (GFAP-ir cells) increased after EtOH exposure and tended to return to normality after abstinence, while cytoplasmic astroglial S100b protein-ir, relative area of MAP-2-ir and Nf-200-ir fibers decreased, and later partially recovered. In the striatum and frontal cortex, nNOS-ir decreased only after abstinence. In conclusion, in the adolescent brain, drinking cessation can partially ameliorate the ethanol-induced morphological changes on neurons and astrocytes but cannot fully return it to the basal state.

Astrocyte-neuron vulnerability to prenatal stress in the adult rat brain.

Chronic activation of the stress response during pregnancy has been shown to be injurious to the development of the offspring. We have previously demonstrated that restraint prenatal stress inflicted during the last week of pregnancy in rats increased dopamine and glutamate receptors in forebrain areas of the adult offsprings. In this study, the same prenatal insult was employed to assess morphological changes in astrocytes and in the dendritic arborization in frontal cortex, striatum, and hippocampus of the adult rat brain. On postnatal day 90, brains were processed for immunocytochemistry using primary antibodies to glial fibrillary acidic protein (GFAP; the main cytoskeletal astroglial protein), S100B protein (an astroglial-derived neurotrophic factor), MAP-2 (a microtubule-associated protein present almost exclusively in dendrites), and synaptophysin (Syn; one major integral protein of the synaptic vesicles membrane). The results show a significant increase in the cell area of GFAP-immunoreactive (-IR) astrocytes, with high levels of S100B protein and a significant decrease in the relative area of MAP-2-IR neuronal processes in prenatally stressed adult rats. The expression of synaptophysin decreased in all areas studied. These results demonstrate that prenatal stress induces a long-lasting astroglial reaction and a reduced dendritic arborization, with synaptic loss in the brain of adult offspring. In addition to the neurochemical alterations previously reported, these morphological changes might be underlying the behavioral and learning impairment previously observed in prenatally stressed rats.