Bioinformatics and in-silico findings reveal medical features and pharmacological targets of biochanin A against colorectal cancer and COVID-19.

Severe mortality due to the COVID-19 pandemic resulted from the lack of effective treatment. Although COVID-19 vaccines are available, their side effects have become a challenge for clinical use in patients with chronic diseases, especially cancer patients. In the current report, we applied network pharmacology and systematic bioinformatics to explore the use of biochanin A in patients with colorectal cancer (CRC) and COVID-19 infection. Using the network pharmacology approach, we identified two clusters of genes involved in immune response (IL1A, IL2, and IL6R) and cell proliferation (CCND1, PPARG, and EGFR) mediated by biochanin A in CRC/COVID-19 condition. The functional analysis of these two gene clusters further illustrated the effects of biochanin A on interleukin-6 production and cytokine-cytokine receptor interaction in CRC/COVID-19 pathology. In addition, pathway analysis demonstrated the control of PI3K-Akt and JAK-STAT signaling pathways by biochanin A in the treatment of CRC/COVID-19. The findings of this study provide a therapeutic option for combination therapy against COVID-19 infection in CRC patients.

The Importance of the Timing of Tocilizumab Administration in Moderate to Severely Ill COVID-19: Single Centered Experience Case series.

One of the main causes of death in COVID-19 is the dysregulation of the host's immune system which leads to cytokine storm, a potentially fatal systemic inflammatory syndrome. Interleukin 6 (IL-6) is a pro-inflammatory cytokine that is produced in response to infections and tissue injuries and is believed to play a pivotal role in the event of a cytokine storm, as signified by its increase in the process. Considering the role of IL-6 as a pro-inflammatory cytokine in the process of cytokine storm in COVID-19, perceiving IL-6 as a therapeutic target could prove to be promising. Tocilizumab is a monoclonal antibody that competitively inhibits the binding of IL-6 to its receptor (IL-6R). The use of IL-6R blocker is recommended for severe COVID-19 patients in the latest therapeutic guideline published by the World Health Organization (WHO), but the timing of the administration has not been specified. While previous studies about the use of tocilizumab in COVID-19 patients have shown various results, these studies do not emphasize on plasma IL-6 levels when deciding the time of tocilizumab administration. In this case series, we present three patients with moderate to severe COVID-19 infections that receive tocilizumab as an adjunct to the standard of care therapy. This case series introduces the novel idea that the timely use of tocilizumab as signified by plasma IL-6 levels in moderate to severe COVID-19 patients could potentially improve overall clinical condition and increase survival rate.

Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice.

CD4+ T cells differentiate into subsets that promote immunity or minimize damage to the host. T helper 17 cells (Th17) are effector cells that function in inflammatory responses. T regulatory cells (Tregs) maintain tolerance and prevent autoimmunity by secreting immunosuppressive cytokines and expressing check point receptors. While the functions of Th17 and Treg cells are different, both cell fate trajectories require T cell receptor (TCR) and TGF-ß receptor (TGF-ßR) signals, and Th17 polarization requires an additional IL-6 receptor (IL-6R) signal. Utilizing high-resolution phosphoproteomics, we identified that both synergistic and additive interactions between TCR, TGF-ßR, and IL-6R shape kinase signaling networks to differentially regulate key pathways during the early phase of Treg versus Th17 induction. Quantitative biochemical analysis revealed that CD4+ T cells integrate receptor signals via SMAD3, which is a mediator of TGF-ßR signaling. Treg induction potentiates the formation of the canonical SMAD3/4 trimer to activate a negative feedback loop through kinases PKA and CSK to suppress TCR signaling, phosphatidylinositol metabolism, and mTOR signaling. IL-6R signaling activates STAT3 to bind SMAD3 and block formation of the SMAD3/4 trimer during the early phase of Th17 induction, which leads to elevated TCR and PI3K signaling. These data provide a biochemical mechanism by which CD4+ T cells integrate TCR, TGF-ß, and IL-6 signals via generation of alternate SMAD3 complexes that control the development of early signaling networks to potentiate the choice of Treg versus Th17 cell fate.

Antibody to Interleukin-6 Receptor Inhibits In Vivo Growth of Human Colorectal Carcinoma Cell Xenografts.

BACKGROUND: Interleukin-6 receptor antibody (IL6R) inhibits colony formation and invasion by colorectal carcinoma (CRC) in vitro. We examined the effect of IL6R antibody on tumor growth of CRC xenografts in vivo. MATERIALS AND METHODS: SW480 cells inoculated subcutaneously into NU/NU mice were treated with anti-IL6R and tumor histology and growth-related signaling were subsequently estimated by hematoxylin and eosin and immunohistochemical staining. RESULTS: Tumor growth was inhibited by anti-IL6R treatment at dosages of both 0.1 and 1.0 mg/kg. Tumor cells had invaded into surrounding tissues in untreated mice, while there was no invasion of tumors in the IL6R antibody-treated mice. The expression of Ki-67, signal transducer and activator of transcription protein 3 (STAT3) and phosphor-extracellular signal-regulated kinase 1 and 2 (ERK1/2) were suppressed in anti-IL6R-treated tumors. CONCLUSION: IL6R antibody inhibited tumor growth and invasiveness in vivo by suppressing the expression of Ki-67, STAT3 and phosphor-ERK1/2. The results imply that the anti-IL6R may be a promising targeted drug for CRC.

Molecular Mechanism of the Anti-Inflammatory Action of Heparin.

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Anti-Interleukin-6 and Janus Kinase Inhibitors for Severe Neurologic Toxicity of Checkpoint Inhibitors.

BACKGROUND AND OBJECTIVES: To describe the marked clinical and biological responses of a targeted treatment with anti-interleukin-6 (IL-6)-receptor antibody and Janus kinase (JAK) inhibitors in a patient with a severe, corticoresistant CNS toxicity of immune-checkpoint inhibitor (ICI) therapy. METHODS: A 58-year-old man was admitted for subacute paraparesis, urinary retention, and ascending paresthesia. He was under treatment with ipilimumab and nivolumab for metastatic melanoma. Spine MRI disclosed multiple T2-hyperintense, contrast-enhancing longitudinally extensive lesions. A diagnosis of ICI-related acute transverse myelitis was made. RESULTS: ICIs were immediately discontinued, and the patient received high-dose glucocorticoids plus 1 session of plasma exchange, but he did not improve. Based on the marked elevation of CSF IL-6 (505 pg/mL), a second-line targeted therapy with anti-IL-6-receptor tocilizumab (8 mg/kg/mo for 3 infusions) plus JAK inhibitor ruxolitinib (50 mg/d) was administered. Patient neurologic status started to improve shortly after, with corresponding radiologic resolution. At 9 months, the patient was able to walk independently, presenting only slight residual disability while remaining in oncologic partial response. DISCUSSION: Our case suggests that some patients with severe, corticoresistant CNS immune-related toxicities of ICIs may benefit from cytokine blockade. Cytokine measurement in serum and CSF might help in selecting patients for personalized treatment strategies.

Kinase activity profiling reveals contribution of G-protein signaling modulator 2 deficiency to impaired regulatory T cell migration in rheumatoid arthritis.

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

Interplay between interleukin-6 signaling and the vascular endothelium in cytokine storms.

Interleukin-6 (IL-6) plays a crucial role in host defense against infection and tissue injuries and is a bioindicator of multiple distinct types of cytokine storms. In this review, we present the current understanding of the diverse roles of IL-6, its receptors, and its signaling during acute severe systemic inflammation. IL-6 directly affects vascular endothelial cells, which produce several types of cytokines and chemokines and activate the coagulation cascade. Endothelial cell dysregulation, characterized by abnormal coagulation and vascular leakage, is a common complication in cytokine storms. Emerging evidence indicates that a humanized anti-IL-6 receptor antibody, tocilizumab, can effectively block IL-6 signaling and has beneficial effects in rheumatoid arthritis, juvenile systemic idiopathic arthritis, and Castleman's disease. Recent work has also demonstrated the beneficial effect of tocilizumab in chimeric antigen receptor T-cell therapy-induced cytokine storms as well as coronavirus disease 2019 (COVID-19). Here, we highlight the distinct contributions of IL-6 signaling to the pathogenesis of several types of cytokine storms and discuss potential therapeutic strategies for the management of cytokine storms, including those associated with sepsis and COVID-19.

Investigation of Fascin1, a Marker of Mature Dendritic Cells, Reveals a New Role for IL-6 Signaling in CCR7-Mediated Chemotaxis.

Migration of mature dendritic cells (DCs) to lymph nodes is critical for the initiation of adaptive immunity. CCR7, a G-protein-coupled receptor for CCL19/21 chemokines, is known to be essential for chemotaxis of mature DCs, but the molecular mechanism linking inflammation to chemotaxis remains unclear. We previously demonstrated that fascin1, an actin-bundling protein, increases chemotaxis of mature mouse DCs. In this article, we demonstrated that fascin1 enhanced IL-6 secretion and signaling of mature mouse DCs. Furthermore, we demonstrated that IL-6 signaling is required for chemotaxis. Blockage of IL-6 signaling in wild-type DCs with an anti-IL-6 receptor α (IL-6Rα) Ab inhibited chemotaxis toward CCL19. Likewise, knockout of IL-6Rα inhibited chemotaxis of bone marrow-derived DCs. The addition of soluble IL-6Rα and IL-6 rescued chemotaxis of IL-6Rα knockout bone marrow-derived DCs, underscoring the role of IL-6 signaling in chemotaxis. We found that IL-6 signaling is required for internalization of CCR7, the initial step of CCR7 recycling. CCR7 recycling is essential for CCR7-mediated chemotaxis, explaining why IL-6 signaling is required for chemotaxis of mature DCs. Our results have identified IL-6 signaling as a new regulatory pathway for CCR7/CCL19-mediated chemotaxis and suggest that rapid migration of mature DCs to lymph nodes depends on inflammation-associated IL-6 signaling.

Interleukin-6 receptor blockade in patients with COVID-19: placing clinical trials into context.

The pleiotropic cytokine interleukin-6 (IL-6) has been implicated in the pathogenesis of COVID-19, but uncertainty remains about the potential benefits and harms of targeting IL-6 signalling in patients with the disease. The efficacy and safety of tocilizumab and sarilumab, which block the binding of IL-6 to its receptor, have been tested in adults with COVID-19-related acute respiratory illness in randomised trials, with important differences in trial design, characteristics of included patients, use of co-interventions, and outcome measurement scales. In this Series paper, we review the clinical and methodological heterogeneity of studies of IL-6 receptor antagonists, and consider how this heterogeneity might have influenced reported treatment effects. Timing from clinical presentation to treatment, severity of illness, and concomitant use of corticosteroids are among the factors that might have contributed to apparently inconsistent results. With an understanding of the sources of variability in these trials, available evidence could be applied to guide clinical decision making and to inform the enrichment of future studies.

Comparison of efficacy between anti-IL-6 receptor antibody and other biological disease-modifying antirheumatic drugs in the patients with rheumatoid arthritis who have knee joint involvement: the ANSWER cohort, retrospective study.

OBJECTIVE: We aimed to investigate the efficacy of anti-IL-6 receptor antibody (aIL-6) and other biologic disease-modifying antirheumatic drugs (bDMARDs), such as TNF inhibitor and CTLA4-Ig in the treatment of rheumatoid arthritis (RA) in patients with knee joint involvement. METHODS: We retrospectively analyzed 1059 treatment courses of patients with RA who visited our hospitals and were treated with bDMARDs. We categorized them into two groups, with or without knee joint involvement. We investigated the clinical disease activity index (CDAI) at baseline and 12 weeks after the initiation of bDMARDs. We compared the improvement of the markers between aIL-6 and other bDMARDs. RESULTS: Treatment with aIL-6 significantly increased ΔCDAI (n = 91, 15.4 ± 1.1; mean ± SEM) in patients with knee joint involvement, compared to other bDMARDs (n = 232, 11.0 ± 0.7) at 12 weeks (P = 0.006). Following the multivariate analysis adjusted by the CDAI levels at baseline, age, gender, concomitant use of methotrexate, and the first use of bDMARDs, ΔCDAI levels were significantly higher in aIL-6, compared to other bDMARDs (P = 0.02). However, there was no significant difference in ΔCDAI improvement between aIL-6 (n = 162, 5.9 ± 0.6) and other bDMARDs (n = 573, 6.2 ± 0.4) in patients without swollen knee joints. ΔCDAI levels were equally increased in patients with shoulder and elbow joint involvement. CONCLUSION: aIL-6 was more effective in the patients with RA and knee joint involvement, compared to other bDMARDs.

Preclinical development of CD126 CAR-T cells with broad antitumor activity.

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive partly due to the restricted use of each CAR construct for specific tumors. Thus, a CAR construct with broad antitumor activity can be advantageous. We identified that CD126 is expressed by many hematologic and solid tumors, including multiple myeloma, lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer, and malignant melanoma among others. CAR-T cells targeting CD126 were generated and shown to kill many tumor cells in an antigen-specific manner and with efficiency directly proportional to CD126 expression. Soluble CD126 did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126 but caused no weight loss or hepatotoxicity in mice. In multiple myeloma and prostate adenocarcinoma xenograft models, intravenously injected CD126 CAR-T cells infiltrated within, expanded, and killed tumor cells without toxicity. Binding of soluble interleukin-6 receptor (sIL-6R) by CAR-T cells could mitigate cytokine release syndrome. Murine SAA-3 levels were lower in mice injected with CD126 CAR-T compared to controls, suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic target for CAR-T cells for many tumors with a low risk of toxicity.

In Silico Studies on Psilocybin Drug Derivatives Against SARS-CoV-2 and Cytokine Storm of Human Interleukin-6 Receptor.

Various metabolites identified with therapeutic mushrooms have been found from different sources and are known to have antibacterial, antiviral, and anticancer properties. Over thousands soil growth-based mushroom metabolites have been discovered, and utilized worldwide to combat malignancy. In this study, psilocybin-mushroom that contains the psychedelic compounds such as psilacetin, psilocin, and psilocybine were screened and found to be inhibitors of SARS-CoV-2 Mprotease. It has been found that psilacetin, psilocin, and psilocybine bind to Mprotease with -6.0, -5.4, and -5.8 kcal/mol, respectively. Additionally, the psilacetin was found to inhibit human interleukin-6 receptors to reduce cytokine storm. The binding of psilacetin to Mprotease of SARS-CoV-2 and human interleukin-6 receptors changes the structural dynamics and Gibbs free energy patterns of proteins. These results suggested that psilocybin-mushroom could be utilized as viable potential chemotherapeutic agents for SARS-CoV-2.

SARILUMAB FOR RECALCITRANT CYSTOID MACULAR EDEMA IN NON-PARANEOPLASTIC AUTOIMMUNE RETINOPATHY.

PURPOSE: To demonstrate a rapid improvement of recalcitrant cystoid macular edema (CME) and perivascular leakage, in a patient with non-paraneoplastic autoimmune retinopathy and autoimmune optic neuropathy after treatment with sarilumab, a human anti-interleukin-6 (IL-6) receptor antibody. METHODS: Observational case report. RESULTS: A 29-year-old woman was diagnosed with non-paraneoplastic autoimmune retinopathy and autoimmune optic neuropathy and followed over 1.5 years. She had recalcitrant CME despite local corticosteroid and immunosuppressive therapy that included azathioprine and adalimumab. Subcutaneous sarilumab was initiated at a dose of 200 mg every 2 weeks. Cystoid macular edema significantly decreased after two injections and resolved after four injections with associated improvement in visual acuity and significant improvement in perivascular leakage on fluorescein angiography. There was a sustained visual and anatomical improvement at 6 months along with mild improvement in electroretinogram responses. The patient tolerated the medication with no side effects. CONCLUSION: Management of CME in non-paraneoplastic autoimmune retinopathy is challenging, and long-term immunosuppression is often employed with varying degrees of success. The improvement in refractory CME and perivascular leakage in this case supports the potential role of an IL-6 inhibitor to treat CME associated with non-paraneoplastic autoimmune retinopathy suggesting the role.

Tocilizumab, an anti-interleukin-6 receptor antibody, efficiently ameliorates persistent joint inflammation in rheumatoid arthritis.

OBJECTIVES: To assess the efficacy of tocilizumab (TCZ) in the treatment of persistent arthritis in patients with rheumatoid arthritis (RA). METHODS: The response to TCZ was evaluated in 304 patients with RA. TCZ treatment was completed after no fewer than 168 consecutive days between 28 May 2008 and 31 July 2019. Efficacy was evaluated using the DAS28-ESR and EULAR response criteria. RESULTS: The mean DAS28-ESR decreased from 4.5 at baseline to 2.0 and 1.5, at 2 months and 1 year after treatment initiation, respectively, and was below 1.5 at 10 years. The retention rate within 1 year was 92.3%. TCZ re-administration to 74 patients with relapsed RA after TCZ withdrawal was also effective. The mean DAS28-ESR decreased from 4.4 at baseline to 1.8 and 1.6 at 2 months and 1 year after retreatment initiation, respectively. The mean swollen joint count decreased from 4.1 in initial TCZ administration and 2.8 in re-administration at baseline to 0.8 and 0.4 at 2 months, respectively. In all patients, good or moderate responses were achieved at least once within 12 months in both initial TCZ administration and re-administration. CONCLUSION: TCZ efficiently ameliorated persistent arthritis in RA, regardless of initial administration and re-administration.

Tocilizumab for the treatment of adult patients with severe COVID-19 pneumonia: A single-center cohort study.

Coronavirus disease 2019 (COVID-19) can lead to a massive cytokine release. The use of the anti-interleukin-6 receptor monoclonal antibody tocilizumab (TCZ) has been proposed in this hyperinflammatory phase, although supporting evidence is limited. We retrospectively analyzed 88 consecutive patients with COVID-19 pneumonia that received at least one dose of intravenous TCZ in our institution between 16 and 27 March 2020. Clinical status from day 0 (first TCZ dose) through day 14 was assessed by a 6-point ordinal scale. The primary outcome was clinical improvement (hospital discharge and/or a decrease of ≥2 points on the 6-point scale) by day 7. Secondary outcomes included clinical improvement by day 14 and dynamics of vital signs and laboratory values. Rates of clinical improvement by days 7 and 14 were 44.3% (39/88) and 73.9% (65/88). Previous or concomitant receipt of subcutaneous interferon-ß (adjusted odds ratio [aOR]: 0.23; 95% confidence interval [CI]: 0.06-0.94; P = .041) and serum lactate dehydrogenase more than 450 U/L at day 0 (aOR: 0.25; 95% CI: 0.06-0.99; P = .048) were negatively associated with clinical improvement by day 7. All-cause mortality was 6.8% (6/88). Body temperature and respiratory and cardiac rates significantly decreased by day 1 compared to day 0. Lymphocyte count and pulse oximetry oxygen saturation/FiO2 ratio increased by days 3 and 5, whereas C-reactive protein levels dropped by day 2. There were no TCZ-attributable adverse events. In this observational single-center study, TCZ appeared to be useful and safe as immunomodulatory therapy for severe COVID-19 pneumonia.

IL-6 as a major regulator of MDSC activity and possible target for cancer immunotherapy.

Myeloid-derived suppressor cells (MDSC) are generated during tumor progression and suppress the anti-tumor functions of T and natural killer (NK) cells. Their enrichment is associated with a bad prognosis and a worse outcome of immunotherapy in cancer patients. The cytokine interleukin (IL)-6 was found to be a crucial regulator of MDSC accumulation and activation as well as a factor, stimulating tumor cell proliferation, survival, invasiveness and metastasis. Accordingly, IL-6 can serve as a negative prognostic marker in cancer. On the other hand, this cytokine is also involved in T cell activation. This review discusses the pleiotropic effects of IL-6 on immune cell populations that are critical for tumor development, such as MDSC and T cells, and summarizes the data on targeting IL-6 or IL-6 receptor (IL-6R) for tumor immunotherapy to block MDSC-mediated immunosuppression in cancer patients.

The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells.

The interleukin-6 (IL-6) membrane receptor and its circulating soluble form, sIL-6R, can be targeted by antibody therapy to reduce deleterious immune signaling caused by chronic overexpression of the pro-inflammatory cytokine IL-6. This strategy may also hold promise for treating acute hyperinflammation, such as observed in coronavirus disease 2019 (COVID-19), highlighting a need to define regulators of IL-6 homeostasis. We found that conventional dendritic cells (cDCs), defined in mice via expression of the transcription factor Zbtb46, were a major source of circulating sIL-6R and, thus, systemically regulated IL-6 signaling. This was uncovered through identification of a cDC-dependent but T cell-independent modality that naturally adjuvants plasma cell differentiation and antibody responses to protein antigens. This pathway was then revealed as part of a broader biological buffer system in which cDC-derived sIL-6R set the in-solution persistence of IL-6. This control axis may further inform the development of therapeutic agents to modulate pro-inflammatory immune reactions.