TNF Protein Production Research Articles

Сytokine expression and production in severe cases of SARS-CoV-2 infection

Most respiratory viral infections proceed in mild form including COVID-19. Gowever, some patients experience severe systemic inflammation, tissue damage, acute respiratory distress syndrome, and cytokine storm with potentially lethal outcomes. The cytokines have been thought to play an important role in immunopathology of viral infection. However, an excessive immune response, manifesting as massive release of pro-inflammatory cytokines, may cause immune damage in the body. Production and release of the cytokines in healthy individuals presumes a significant balance of inflammatory and homeostatic factors. Meanwhile, in the case of COVID-19 disease, uncontrolled increased production of cytokines often occurs with fatal consequences for patients. The aim of this work was to study the level of IL-1β, IL-18 and TNFα gene expression, as well as production of these cytokines at the level of mucous membranes of the upper respiratory tract, in particular in oral cavity, in patients with severe COVID-19 disease.
 The present study included patients who recovered from severe COVID-19. The control group consisted of conditionally healthy individuals. Expression levels of the IL-1β, IL-18, and TNFα genes were determined by RT-PCR. The levels of IL-1β, IL-18 and TNFα protein production were determined by multiplex enzyme immunoassay.
 The expression levels of IL-1β, IL-18 were reduced at the onset of the disease, as well as in the midpoint of the COVID-19 disease, but increased on the 30th day. The protein production of these cytokines was also reduced in the first days from the onset of the disease. The levels of pro-inflammatory TNFα cytokine was high at the onset of the disease. The level of TNFα production at the onset of the disease was also higher relative to the control group. Subsequently, the TNFα gene expression levels decreased upon progression of the disease.
 Thus, the increased expression level of pro-inflammatory cytokines may be explained by the fact that the S protein of the SARS-CoV-2 virus induces increased expression of these cytokines in human monocytes. Meanwhile, appropriate protein levels remain low, especially on day 1 from the onset of the disease. Thus, one may conclude that the virus triggers pyropotosis, however, within 15-30 days from the onset of the disease, when viral replication is already minimal.

Cyclic AMP signaling enhances lipopolysaccharide sensitivity and interleukin-33 production in RAW264.7 macrophages.

While it has been suggested that IL-33 plays pathogenic roles in various disorders, the factors that stimulate IL-33 production are poorly characterized. In the present study, the effect of cyclic adenosine monophosphate (cAMP) signaling on IL-33 production in RAW264.7 macrophages in response to various doses of LPS was examined. High-dose LPS treatment induced IL-33 and TNF protein production in RAW264.7 macrophages. In contrast, low-dose LPS failed to induce IL-33 production while significantly inducing TNF production. In the presence of the membrane-permeable cAMP analog 8-Br-cAMP, low-dose LPS induced vigorous IL-33 production. This phenomenon was consistent with amounts of mRNA. Similarly, the cAMP-increasing agent adrenaline also enhanced the sensitivity of RAW264.7 macrophages to LPS as demonstrated by IL-33 production. The protein kinase A (PKA) inhibitor H89 blocked the effects of 8-Br-cAMP and adrenaline on IL-33 production, suggesting that PKA is involved in IL-33 induction. Taken together, cAMP-mediated signaling pathway appears to enhance the sensitivity of RAW264.7 macrophages to LPS with respect to IL-33 production. Our findings suggest that stress events and the subsequent secretion of adrenaline enhance macrophage production via IL-33; this process may be associated with the pathogenesis of various disorders involving IL-33.

Increased TNF expression in CD43 ++ murine blood monocytes

Monocyte heterogeneity has been studied extensively in man but only recently tools have been developed to study blood monocyte populations in the mouse. We have used the MacGreen mouse model, which expresses the green fluorescent protein under the control of the promoter of the murine M-CSF receptor (CSF1 receptor, c-fms). Since both monocytes and granulocytes show GFP expression in this model the latter cells were excluded by staining with the Ly6G granulocyte marker. GFP + Ly6G − blood monocytes were found to account for an average of 246 ± 121 cells/μl in these mice. These monocytes can be subdivided into CD43 + GR-1 + cells and CD43 ++ GR-1 − cells, with the latter cells accounting for 140 ± 77 cells/μl, i.e. about 60% of all blood monocytes. After intraperitoneal injection of lipopolysaccharide (LPS) both blood monocyte subpopulations were depleted. The same was true after intranasal infection with Streptococcus pneumoniae but here the CD43 ++ subpopulation was preferentially reduced to 4 cells/μl. For the study of TNF expression cells were stimulated in vitro with LPS from Salmonella abortus equi in the presence of Brefeldin A followed by intracellular staining and multicolor flow cytometry. Over a dose range of 10–100 ng LPS/ml, TNF protein production was significantly higher in the CD43 ++ monocyte subset. At 1000 ng LPS/ml 90% of all CD43 ++ monocytes stained positive for TNF and in terms of fluorescence intensity TNF was 5-fold higher compared to the CD43 + monocytes. These data indicate that the murine CD43 ++ monocyte subset exhibits features of pro-inflammatory monocytes and is functionally homologeous to the human CD14 +CD16 + monocytes.

The effect of beta‐glucan pretreatment on TNF production in vivo

The soluble beta‐glucan preparation PGG‐glucan has been shown to prime antimicrobial functions, including increasing the oxidative burst response, microbicidal activity, and the chemotaxis of leukocytes, without eliciting the production of proinflammatory cytokines. In vitro experiments using primary macrophages and macrophage‐like cell lines have demonstrated that pretreatment with PGG‐glucan can reduce the production of TNF message and protein upon LPS stimulation. The purpose of this study was to examine the effect of PGG‐glucan pretreatment on LPS‐induced TNF protein production in vivo. The hypothesis is that PGG‐glucan pretreatment will result in a reduction of LPS‐induced TNF‐protein levels. To test this hypothesis, CD‐1 mice were pretreated with a single dose of PGG‐glucan 40 hours prior to stimulation with LPS. Following LPS stimulation, serum TNF protein levels were measured by ELISA. Our data suggest that PGG‐glucan pretreatment of mice can attenuate the LPS‐induced TNF production in serum. These results indicate that PGG‐glucan can stimulate cellular immune function while inducing suppression of proinflammatory cytokines in vitro and in vivo. These data and future studies may identify a pharmacological intervention that allows cells of the innate immune system to be primed while not overproducing inflammatory cytokines.Supported by NIHGM66194

HDAC3, a Class I Histone Deacetylase, Interacts with the P38 Pathway and Suppresses MAPK11-Mediated ATF-2 Activation and TNF Gene Expression.

During inflammatory events, the induction of immediate-early genes, such as tumor necrosis factor α (TNF), is regulated by signaling cascades including the JAK/STAT, NF-kB, and the p38 mitogen-activated protein kinase (MAPK) pathways, which result in phosphorylation-dependent activation of transcription factors. We observed the direct interaction of HDAC3, a class I histone deacetylase, with MAPK11 (p38 beta isoform) by west-western-based screening analysis, pull-down assay and two-hybrid system analysis. Results further indicated that HDAC3 decreases the MAPK11 phosphorylation state and inhibits the activity of the MAPK11-dependent transcription factor ATF-2. LPS-mediated activation of ATF-2 was inhibited by HDAC3 in a time- and dose-dependent manner. Inhibition of HDAC3 expression by RNA interference resulted in increased ATF-2 activation in response to LPS stimulation. In agreement with decreased ATF-2 transcriptional activity by HDAC3, HDAC3 repressed TNF gene expression and TNF protein production observed in response to LPS stimulation. Therefore, our results indicate that HDAC3 interacts directly and selectively with MAPK11, represses ATF-2 transcriptional activity and acts as a regulator of TNF gene expression in LPS stimulated cells, especially in mononuclear phagocytes.

Histone deacetylase 3, a class I histone deacetylase, suppresses MAPK11-mediated activating transcription factor-2 activation and represses TNF gene expression.

During inflammatory events, the induction of immediate-early genes, such as TNF-alpha, is regulated by signaling cascades including the JAK/STAT, NF-kappaB, and the p38 MAPK pathways, which result in phosphorylation-dependent activation of transcription factors. We observed the direct interaction of histone deacetylase (HDAC) 3, a class I histone deacetylase, with MAPK11 (p38 beta isoform) by West-Western-based screening analysis, pull-down assay, and two-hybrid system analysis. Results further indicated that HDAC3 decreases the MAPK11 phosphorylation state and inhibits the activity of the MAPK11-dependent transcription factor, activating transcription factor-2 (ATF-2). LPS-mediated activation of ATF-2 was inhibited by HDAC3 in a time- and dose-dependent manner. Inhibition of HDAC3 expression by RNA interference resulted in increased ATF-2 activation in response to LPS stimulation. In agreement with decreased ATF-2 transcriptional activity by HDAC3, HDAC3-repressed TNF gene expression, and TNF protein production observed in response to LPS stimulation. Therefore, our results indicate that HDAC3 interacts directly and selectively with MAPK11, represses ATF-2 transcriptional activity, and acts as a regulator of TNF gene expression in LPS-stimulated cells, especially in mononuclear phagocytes.

The in vivo effects of tumour necrosis factor blockade on the early cell mediated immune events and syndrome expression in rat adjuvant arthritis.

Anti-TNF therapy is effective in rheumatoid arthritis (RA); however, its mechanisms of action are incompletely understood. T cell-driven mechanisms are thought to play an important role in RA and the effects of TNF blockade on these mechanisms are unclear. Adjuvant arthritis (AA) is a T cell dependent model of inflammatory arthritis. The aims of this study were to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph nodes (ILN) in early arthritis. AA was induced in male DA rats. Rats received either 3 mg/kg or 10 mg/kg PEG sTNF-RI at days 0, 2 and 4 postinduction or 10 mg/kg anti-TNF antibody on day of arthritis induction. Control rats received either saline or normal sheep serum. Paw volume was assessed every 3-4 days. Rats were sacrificed on days 0, 6, 13 and 21 postinduction. Ankles were removed for quantitative radiology and histology. Synovium and ILN were removed for cell culture and to determine mRNA expression of cytokines using semiquantitative RT-PCR. TNF and IFN-gamma protein production was measured using a bioassay and an ELISA. TNF blockade did not suppress mRNA expression of T cell cytokines in the ILN of rats in the early phase of AA, suggesting ongoing T cell activity. TNF protein production by ILN cells in culture was reduced in PEG sTNF-RI treated rats, although mRNA expression was increased in the ILN prior to culture. Early administration of PEG sTNF-RI did not attenuate AA, in contrast to an anti-TNF antibody, which suppressed disease. A shorter half-life for the PEG sTNF-RI compared with the anti-TNF antibody or the development of anti-PEG sTNF-RI antibodies may account for these results.

In vivo treatment with the fungal metabolite gliotoxin suppresses intestinal inflammation in dextran sulphate sodium [DSS]-induced colitis

Back~,round: NF-kB is a transcription factor, which plays a central role in immune and inflammatory responses. Gliotoxin, a toxic metabolite produced by pathogenic fungi, inhibits the activation of NF-kB in vitro (Pahl, HL et aL; J.Exp. Med. 183: 1829-40; 1996), which indicates antiinfiammatory properties. We therefore investigated whether gliotoxin was able to downregulate inflammatory cytokine production in vitro and intestinal inflammation in the mouse model of acute DSS-induced colitis. Methods: In vitro experiments: Gel mobility shift assays were performed using nuclear extracts of EL-4 cells, stimulated with PMA plus IL-1 in the presence or absence of gliotoxin. RAW-264.7 cells were stimulated with 10pg LPS and different amounts of gliotoxin (0.1-10pg/ml) for 8 hours. Supernatants were collected and secreted TNF-a and IL-6 were measured by ELISA. In vivo experiments: Colitis was induced by oral administration of 5% DSS dissolved in the drinking water given for 7 days to Balb/c mice. Treatment with intraperitoneal administration of gliotoxin (20 or 2x20 pg/mouse daily for 5 days) or saline was performed in 2 independent experiments (n=5/group, 2x20 ~g Gliotoxin only 1 Exp.) and was begun on day 3 of DSS application Mice were killed and evaluated on day 8. Extent of colonic inflammation was estimated histologically [max.grade 4] and tissue expression of IL 1 and TNF was analyzed by RT-PCR. Colonic IL-6 protein production was measured by ELISA. Results: Gliotoxin inhibited PMA plus IL-1 stimulated activation of NF-kB nuclear translocation. Furthermore, Gliotoxin downregulated IL-6 and TNF protein production in a dose dependent manner. In vivo a significant amelioration of the histological score was achieved with gliotoxin treatment compared to placebo treated animals [ Mean + SEM: 2.8 -+ 0.3 DSS/Placebo; 1.4 + 0.3 DSS/Gliotoxin 20pg*; 0.4 + 0.1 DSS/Gliotoxin 2x20pg*; 0.6-+ 0A H20/placebo; *p<0.01; n=10 except n=4 2x20 DSS/gliotoxin]. Giving 2x20 ~g gliotoxin resulted in death of one mouse. Gliotoxin treatment suppressed in vivo mRNA expression of IL-1 and TNF-a and downregulated IL-6 protein production in colonic specimens. Conclusion: The fungal metabolite gliotoxin is a powerful immunsuppressive agent, although when administered systemically at higher doses a toxic effect can not be excluded. Further studies of this agent as a potential therapeutic alternative in inflammatory bowel disease are warranted.

The Immunomodulatory Effects of the Herbicide Propanil on Murine Macrophage Interleukin-6 and Tumor Necrosis Factor-α Production

Intraperitoneal (ip) exposure to propanil (3,4-dichloropropionanilide) has previously been shown to affect macrophage cytotoxicity. In this study, we compared the immunotoxic effects of propanil, after different routes ofin vivoadministration, on cytokine production by thioglycollate-elicited peritoneal macrophages. C57B1/6 mice were treated with either vehicle or 200 mg/kg propanil ip, or with vehicle, 40, or 400 mg/kg propanil orally. Three or 7 days later,ex vivoproduction of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by macrophages after lipopolysaccharide (LPS) stimulation was determined. Both oral and ip propanil exposure resulted in up to a 60–70% reduction in IL-6 and TNF-α production by the LPS-stimulated macrophages, depending on the route, postexposure time, and dose of propanil administered. Oral exposure to propanil also caused splenomegaly and thymic atrophy in animals in much the same manner as animals exposed via the ip route.In vitroexposure to propanil also significantly reduced macrophage cytokine production. Thioglycollate-elicited macrophages from normal mice were cultured in the continuous presence of 0, 10, or 20 μmpropanil plus LPS. This exposure caused a significant reduction in IL-6 and TNF protein production by these macrophages in a concentration-dependent manner. Northern blot analysis demonstrated that the message levels of these cytokines were reduced by approximately the same percentage as the protein levels in propanil-treated macrophages, indicating a possible transcriptional or pretranscriptional target(s) for propanil.

IL-11 enhances survival and decreases TNF production after radiation-induced thoracic injury.

We hypothesized that IL-11 would protect against radiation-induced thoracic injury. To test this hypothesis, we compared the survival of rIL-11 and vehicle-treated control mice after 25 Gy of thoracic irradiation, and initiated studies to elucidate the mechanism of the observed protection. This dose of radiation killed 50% of the control mice during the first 2 wk after irradiation. In contrast, the s.c. administration of rIL-11 resulted in significant radioprotection with 89% of the rIL-11-treated animals surviving the study interval (p < 0.001). This radioprotection was at least partially specific for normal thoracic structures since rIL-11 did not alter the development or radiosensitivity of EMT6 tumor cells growing as lung metastases. TNF mRNA was not detected in normal lungs but was impressively induced after thoracic irradiation. Treatment with rIL-11 abrogated this increase. Parallel in vitro studies demonstrated that rIL-11 inhibits LPS and radiation-induced macrophage TNF protein production and mRNA accumulation. These studies demonstrate that rIL-11 reduces the mortality following thoracic irradiation, without enhancing the development or diminishing the radiosensitivity of pulmonary metastatic tumors. They also demonstrate that rIL-11 inhibits both radiation-induced TNF mRNA expression in vivo and macrophage TNF protein production and mRNA accumulation in vitro, suggesting that the radioprotective effects of rIL-11 may be mediated, at least in part, via the modulation of TNF production.

Cyclosporine A Blocks Induction of Tumor Necrosis Factor-α in Human B Lymphocytes

The effects of cyclosporin A (CSP) on tumor necrosis factor-alpha (TNF-α) RNA levels and protein production in human B cells and a B cell line were studied. The ability of CSP to block induction of RNA was compared to its inhibition of protein production in response to anti-IgM, phorbol ester (PMA) and platelet-activating factor (PAF). PAF is a phospholipid which has recently been found to activate human B cells. CSP blocks PAF-induced TNF-α RNA from the Ramos cell line, as well as inhibiting the enhancement of TNF protein production from both freshly isolated and Ramos B cells. CSP also blocks anti-Ig induced TNF-α RNA and protein but does not inhibit PMA-induced TNF-α. We conclude that B cells, like T cells, have CSP-independent and CSP-dependent signaling pathways and that PAF signaling is dependent upon a CSP-sensitive factor.

Pyrrolidine dithiocarbamate inhibits NF-kappa B mobilization and TNF production in human monocytes.

The human TNF promoter contains four potential nuclear factor-kappa B (NF-kappa B)-binding sites, with the strongest binding seen for the -605 motif. Nuclear extracts from unstimulated cells of the human monocytic cell line, Mono Mac 6, contain one specific binding protein (complex II), consistent with a constitutive p50 homodimer. Stimulation of Mono Mac 6 cells with LPS will increase complex II and will strongly induce a second specific complex (complex I), which represents the p50/65 heterodimer. Treatment of Mono Mac 6 cells with pyrrolidine-dithiocarbamate (PDTC) at 300 microM will block the LPS-induced complex I almost completely and will reduce complex II to the constitutive level. Binding activity of other nuclear factors that recognize the SP-1 and c/EBP motifs of the human TNF promoter is not affected by such treatment. Northern blot analysis demonstrates that PDTC treatment will strongly reduce LPS-induced TNF transcripts. Secreted TNF protein as detected in the Wehi 164S/ActD bioassay and in a sandwich immunoassay was similarly reduced by PDTC. Kinetic analyses show that after LPS stimulation, NF-kappa B will peak at 1 h, TNF transcript prevalence at 2 h, and TNF protein at 4 h. PDTC did not shift this response to LPS to a later time, but suppressed NF-kappa B mobilization, TNF transcripts, and TNF protein over the entire 8-h observation period. Analysis of freshly isolated, LPS-stimulated blood monocytes showed a similar blockade of NF-kappa B. Furthermore, in these primary cells, induction of TNF transcripts, as determined by Northern blot analysis and by quantitative polymerase chain reaction, was prevented by PDTC as was TNF protein production. These data show that dithiocarbamates can profoundly affect cytokine expression and suggest that NF-kappa B is involved in LPS-induced TNF gene expression in human monocytes.