Of note, these genes were also upregulated by BSO alone. Interestingly, among the genes upregulated by LPS and decreased by BSO Group 2 , we found genes important in innate immunity and inflammation il1b, Irf7, Irf9, Mx2, Oas2, Oas3, Ptgs2 , as well as the secreted l -phenylalanine oxidase, il4i1.

None of these genes were affected by BSO alone. The list of the top 15 transcripts most affected by BSO among those downregulated by LPS is available as Table S1 in Supplementary Material. The general functions of the four groups of genes differentially regulated by GSH depletion and LPS were then analyzed using DAVID to identify the enriched GO:BP categories and KEGG pathways For this purpose, we combined the list of differentially expressed genes at 2 and 6 h.

Figure 4 shows the KEGG and GO:BP categories overrepresented in each of the four groups. Only categories that included three or more genes are shown. The analysis confirms that Group 1 included genes associated with the response to oxidative stress.

Group 2 included genes associated with immune response, inflammation, and antiviral host defense such as interferon IFN and toll-like receptor TLR signaling. Figure 4. Enriched functional categories in the four groups of genes differentially regulated by LPS and BSO.

The lists of genes in the four groups at 2 and 6 h were combined and the overrepresented GO biological process GO:BP categories white bars and KEGG pathways gray bars were obtained by DAVID analysis.

All categories identified by DAVID for Groups 1—4 are reported. BSO, buthionine sulfoximine; GO:BP, gene ontology biological process; LPS, lipopolysaccharide.

Among the genes whose expression was inhibited by LPS Groups 3 and 4 , only few mapped to some functional category. Group 3 included genes associated with xenobiotics metabolism such as GSH transferases mu 1—4 and cytochrome P The only genes that were part of a functional category in Group 4 were C1q components.

To identify possible common molecular mechanisms responsible for the differential regulation by BSO of the LPS-induced genes in Groups 1 and 2, we performed an unbiased analysis for the overrepresented TF-binding sites using oPOSSUM software In Group 1 Figure 5 A , the TF results in the highest Fisher score and a high number of target genes was NFE2L2 nrf2 , whose main function is the response to oxidative stress, thus confirming the results obtained with DAVID.

In Group 2 Figure 5 B , the TF that had the highest score was NF-kB with its various subunits. Figure 5. The number in parentheses indicates the number of transcripts that map to each TF. TF, transcription factor. We thus manually searched our dataset for the expression of known NF-kB target genes.

However, only 8 out of 87 at 2 h and 4 out of at 6 h were downregulated by BSO. Thus, because only a very small percentage of NF-kB target genes induced by LPS are in Group 2 downregulated by BSO , we could rule out that BSO acts simply by downregulating NF-kB. Microarray results were validated by RT-qPCR for 11 genes Figures 6 and 7.

Ten genes were selected from Groups 1 or 2, at 2 and 6 h. Since we have a specific interest in this gene, it was selected for validation by RT-qPCR. Figure 6. PCR validation of the microarray data at 2 h. Data are expressed as fold change vs one of the respective control samples. For each experimental group, the mean is also shown.

PCR, polymerase chain reaction. Figure 7. PCR validation of the microarray data at 6 h. We performed validation in two sets of samples: one with the same RNA used for the microarray experiment qPCR1 and one with RNA from an entirely independent experiment qPCR2.

For all 11 genes tested, PCR confirmed the differential expression detected by microarray analysis both at 2 and 6 h Figures 6 and 7 , respectively.

In the second experiment, at 2 h results were confirmed for five out of seven genes, including il1b, Irf9, Mx2, Il4i1, and Srxn1; at 6 h, three genes out of four were validated including Prdx1, Nos2, and Slc7a Interestingly, by RT-qPCR we could find a statistically significant inhibitory effect of BSO on LPS-induced Nos2, which did not pass the correction for multiple comparisons; this is not surprising, since the false discovery rate correction, being more conservative, can generate false negatives.

We decided to show the more reliable results obtained in the two independent experiments assayed by PCR Figure 7 ; however, for consistency, Nos2 was not included in any subsequent analysis functional analysis, TF analysis , and is not listed in File S1 in Supplementary Material.

We wondered whether the GSH requirement in the induction of genes in the IFN response pathway in Group 2 was biologically relevant. Therefore, we investigated the effect of LPS on PR8 influenza virus infection in RAW cells in which GSH had been depleted by BSO.

As shown in Figure 8 , when cells were infected with PR8, LPS reduced infection, in terms of intracellular viral protein production; influenza nucleoprotein NP, the most expressed among the viral proteins was significantly decreased in cells pretreated with LPS.

However, the effect of LPS was not observed in GSH-depleted cells. Although, as reported previously, BSO alone increased NP production 21 , the treatment with both LPS and BSO induced a further significant increase.

Figure 8. LPS activation of antiviral innate immunity is dependent on GSH. A Western blot for influenza virus proteins in RAW cells infected with PR8 or uninfected, after LPS treatment, with and without GSH depletion. β-Actin was used as loading control. B Levels of NP viral protein in RAW cells pretreated with LPS, with and without GSH depletion.

GSH, glutathione; LPS, lipopolysaccharide; NP, nucleoprotein. We next asked the question whether the inhibitory effect of GSH on Group 1 genes, as revealed by the upregulation by BSO, might be due to its ROS-scavenging antioxidant action. To answer this, we first investigated whether the induction of Group 1 genes by LPS was inhibitable by the thiol antioxidant NAC.

Second, to investigate whether ROS generation induced by LPS could have a role in the induction of Group 1 genes, we asked whether a ROS-generating agent menadione would reproduce the effect of LPS. As shown in Figure 9 , NAC did not alter the induction of selected Group 1 genes Srx1, Prdx1, Slc7a On the other hand, all these genes were induced by menadione alone.

Figure 9. Effect of NAC and menadione on Group 1 left and Group 2 right genes. Menadione Men was added at 10 µM for 2 h. Gene expression was measured by qPCR. Data are expressed as fold change vs one of the control samples, and are the mean ± SD of six biological replicates from two independent experiments.

LPS, lipopolysaccharide; NAC, N -acetyl- l -cysteine; qPCR, quantitative polymerase chain reaction. The same experimental framework was used to study the relevance of the ROS scavenging properties of GSH in its permissive role for the induction of Group 2 genes.

Opposite to what observed with Group 1 genes, menadione by itself was unable to regulate the expression of any of Group 2 genes measured. This study supports the view that endogenous GSH plays a pivotal role for the establishment of the innate immune responses to viruses, possibly acting as a signaling molecule with a mechanism different from simple scavenging of ROS.

The fact that the vast majority of transcripts were unaffected by BSO is also an indirect confirmation that, within the concentrations and incubation times used, BSO does not have significant toxic or non-specific effects. The observation that GSH depletion does not exacerbate the transcription of inflammatory genes, at least in our experimental conditions, might seem at variance with the existing literature starting from pioneering paper by Schreck et al.

However, most of that evidence is based on in vitro or in vivo experiments using exogenously administered thiol antioxidants or pro-oxidants. What our data do not support is the extrapolation of evidence from those experiments to the conclusion that GSH is an endogenous anti-inflammatory molecule through its ROS-scavenging activity.

In fact, previous reports noted that exogenous GSH or its precursor NAC inhibits the production and expression of TNF, IL-6, and IL-8 by LPS-stimulated macrophages in the absence of any significant change in intracellular GSH The results reported here are also in agreement with our previous studies where we observed that there are more H 2 O 2 -induced genes that require GSH for their upregulation than genes whose induction by H 2 O2 is exacerbated by GSH depletion Interestingly, in that study using human monocytic cells, many of the H 2 O 2 -induced genes for which GSH had a facilitatory role were related to immunity In addition, the only LPS-induced transcripts mapping to innate immunity in their functional annotation were inhibited, rather than upregulated, by GSH depletion Group 2 genes.

Not only innate immunity genes in Group 2 require GSH for their induction but also they were not induced by ROS alone using menadione as a ROS-generating chemical and their LPS induction was not inhibited by NAC, ruling out the possibility that ROS act as signaling molecules in their induction by LPS.

The only exception was il1b whose LPS induction was inhibited by NAC but was also inhibited by GSH depletion, suggesting that GSH is important for IL-1b induction by LPS but possibly not through an antioxidant mechanism because i exogenous NAC and endogenous GSH appear to have an opposite role, and ii an oxidant alone does not induce IL-1b expression.

In line with these findings, it has been shown that molecules altering intracellular thiol content with different mechanisms i. The innate immune response is also important for antiviral defense and activation of TLR4 leads to induction of antiviral proteins including IFNs and IFN-related genes 27 , 28 such as MxA and Oas 29 , Our data, although obtained in a model where infectivity was low, suggest that GSH is important for the activation of an antiviral response.

This happens without affecting inflammatory genes, except for IL-1b whose induction was also facilitated by the presence of GSH. There is evidence for a fine-tuning of TLR signaling 31 , and these data indicate that GSH may be important in directing it toward specific small patterns of genes implicated in host defense rather than toward those responsible for the inflammatory response, as outlined in Figure Figure GSH fine-tuning of TLR4 signaling.

GSH orients the TLR4-mediated changes in gene expression profile toward activation of host defense. GSH, glutathione; LPS, lipopolysaccharide; TLR4, toll-like receptor 4.

The behavior of genes in Group 1 is what one would expect. They include enzymes for GSH synthesis and antioxidant enzymes such as Prdx1, Srxn1, and Hmox. All these genes map to nrf2, a master regulator of redox homeostasis Their regulation by BSO is in accordance with the hypothesis that endogenous GSH acts as an ROS scavenger because menadione induces their expression.

However, NAC did not inhibit their induction by LPS, suggesting that LPS induces nrf2 target gene expression independently of the increase in ROS production.

This agrees with a recent study by Cuadrado et al. showing that LPS can activate nrf2 via the small GTPase RAC1, independently of ROS In this picture, endogenous GSH might be important through other mechanisms than just scavenging ROS.

In fact, nrf2 activation is dependent on oxidation of its redox sensor, keap1. Several studies have indicated that activation of nrf2 by administration of electrophilic compounds has an anti-inflammatory effect and decreases LPS-induced transcription of other NF-kB target genes, including TNF, IL-1b, and IL-6, in RAW cells 35 , However, as mentioned earlier, in our experimental conditions in which nrf2 was likely activated by GSH depletion, as suggested by the increased expression of nrf2 target genes, we have not observed an effect on any inflammatory cytokine other than IL-1b.

Once again, the difference might be that we did not use exogenous electrophiles to induce nrf2. This highlights one point that is often overlooked. GSH is not just an antioxidant that participates in ROS elimination either via its direct ROS scavenging activity or as a substrate for GSH peroxidases but, like any other thiol including NAC, is also a reducing agent, as well as GSSG is a thiol oxidizing agent.

Therefore, these two molecular species, GSH and GSSG, can regulate biological pathways in a redox-dependent manner, independently of ROS scavenging.

In fact, protein glutathionylation is a major mechanism of redox regulation of immunity 10 , 37 , affecting the function of key proteins including NF-kB 38 , STAT3 39 , PKA 40 , TRAF3, and TRAF6 41 , as well as participating in the release of danger signals 42 , However, in this experimental model, the induction of host defense genes in Group 2 at least those shown in Figure 7 , il1b, Mx2, and Irf9 is inhibited by BSO, evidencing the need for GSH, but is not amplified by NAC, suggesting that scavenging LPS-induced ROS is not the main mechanism of action of endogenous GSH.

The finding that several genes that are important for the antiviral response, mostly part of IFN signaling pathways, including the antiviral proteins Oas and Mx2, require GSH for optimal induction by LPS adds knowledge to previous findings, indicating that GSH can inhibit viral infection 44 , 45 and that viral infection causes release of glutathionylated thioredoxin and Prdx There is a large body of evidence showing the importance of GSH in immunity, including antiviral immunity 47 , but so far this was ascribed to its action as ROS scavenger to inhibit oxidative stress.

The present study indicates that GSH has other important signaling roles independently of protection from oxidative stress, and its action may not be vicariated by another thiol antioxidant. However, to understand the validity of our conclusions to other models, one needs to bear in mind the limitations of this study that is investigating mRNAs in a cell line.

Future studies will need to measure the proteins of interest for instance, IL-1b to see whether the changes observed at the level of transcripts are reflected in changes in protein levels. To generalize the relevance of this mechanism, the observation will need to be confirmed in primary cells, including human cells, and possibly in vivo.

MD, PC, MM, IC, LC, FP, and KA performed experiments. AH, PG, KA, LC, MM, FP, and AP designed and supervised experiments. MD, PG, MM, FP, and PC wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This work was supported by a fellowship program from Istituto Pasteur Italia——Fondazione Cenci Bolognetti to PC , PRIN CUP grant number B to AP , and RM Phillips Trust to PG. File S1. Transcripts in bold are those also significantly affected by BSO alone BSO vs control, with a cutoff of FC 1.

The log 2 -transformed gProcessed signals of the three biological replicates are shown. The FC between the two groups indicated is expressed as log 2 ratio. Limit alcohol consumption. We all know that alcohol consumption affects our liver. Cell death in the liver may be exacerbated by a deficiency in antioxidants, including glutathione.

This can lead to fatty liver disease. A study reported that glutathione is very effective when given to people with fatty liver disease intravenously, in high doses.

Eat an antioxidant-rich diet. Sulfur-rich foods, like: dietary proteins, such as beef, fish and poultry. cruciferous vegetables like broccoli, Brussels sprouts, cauliflower, kale, watercress and mustard greens can all boost antioxidant levels in the body.

Take a glutathione supplement. One of my most frequent supplement recommendations is liposomal glutathione. Almost everybody can benefit from a daily glutathione supplement.

Take Glutathione Boosting Supplements. Many other vitamins and minerals contribute to glutathione production in the body, including: Curcumin, N-acetylcysteine, Selenium, Silymarin, Vitamin C, and Vitamin E.

Unless a live host comes along, they will eventually die. While not technically alive, viruses will not survive for long unless they find a host cell within a person, animal, or plant to live in.

Bacteria on the other hand, such as strep throat, is a sickness caused by Salmonella and Streptococcus bacteria are alive as a single cell organism, which can live on its own. Antigens are a molecular pattern found on the surface of pathogens, toxins, chemicals, and pollens made of proteins, polysaccharides, lipids or nucleic acids.

For each specific pathogen and disease it causes, the Antigen pattern is unique. The more scientific terminology is 'science pathogen-associated molecular patterns PAMPs. Antigens give the immune system a warning sign that a new foreign microbe has been found in the body and must deal with the potential threat.

Components of an antigen called epitopes are the regions where the antigen and the antibody connect on the pathogenic cell, a bit like a space docking station.

Image above: Illustration design of antibodies. B Cells B lymphocytes are white blood cells that produce antibodies to fight invader microbes or toxins pathogens after exposure to an antigen.

Invading microbes are produced from internal or external sources, i. the air we breathe, food we eat, or internal stress on the body. Antibodies contain a paratope, a small region at the antibody's tip that binds to the antigen epitope with high affinity.

This binding mechanism, a bit like a lock epitope and key paratope , or two puzzle pieces with the same unique pattern, enables them to interlock together. This process allows for the antibody B-cells to track the pathogen and prepare for a defence.

B-cells are each programmed to make only one specific antibody which can take up to several days to develop. For this reason, we can sometimes feel ill for several days as the army of antibodies builds. Humans generate billions, if not trillions of different antibodies over a lifetime, each capable of binding to a distinct epitope the docking station of an antigen.

The antibody recruits other defensive molecules and immune cells, like T-cells in the bloodstream, to make their way to the now marked invader. They all work together to neutralise the infection-causing pathogen. Once killed, it is then engulfed and digested by macrophage cells.

Macrophages are specialist, quite large, white blood cells which scavenge the body looking to remove dying or dead foreign bodies pathogens. The term 'macrophage' is formed from the Greek terms "makro" meaning big, and "phagein" meaning eat.

After the first exposure to a new microbe, the immune system learns and keeps a record of everyone it has ever defeated. If it appears again, it knows to defend against it, this time more swiftly, preventing you from feeling ill. Image above: T-Cell Immune Response.

T-cells are like the special armed services SAS , where a strong reaction is needed to deal with a specific foreign antigen for maintaining effective immunity. T-cells use cytokines cells as messenger molecules which aid cell to cell communication for ramping up its response to move cells towards sites of inflammation, infection and trauma.

There are two main T-Cell role types, 'Helper cells', which help activate B cells to produce antibodies. Or they can act as "Natural Killer Cells" NK , which directly attack and kill cells already infected by a foreign invader Pathogen.

When T-cells become active, they produce reactive oxygen species ROS.