Randomized double-blind clinical study in patients with COVID-19 to evaluate the safety and efficacy of a phytomedicine (P2Et).

Background: It has been proposed that polyphenols can be used in the development of new therapies against COVID-19, given their ability to interfere with the adsorption and entrance processes of the virus, thus disrupting viral replication. Seeds from Caesalpinia spinosa, have been traditionally used for the treatment of inflammatory pathologies and respiratory diseases. Our team has obtained an extract called P2Et, rich in polyphenols derived from gallic acid with significant antioxidant activity, and the ability to induce complete autophagy in tumor cells and reduce the systemic inflammatory response in animal models. Methods: In this work, a phase II multicenter randomized double-blind clinical trial on COVID-19 patients was designed to evaluate the impact of the P2Et treatment on the clinical outcome and the immunological parameters related to the evolution of the disease. The Trial was registered with the number No. NCT04410510*. A complementary study in an animal model of lung fibrosis was carried out to evaluate in situ lung changes after P2Et in vivo administration. The ability of P2Et to inhibit the viral load of murine and human coronaviruses in cellular models was also evaluated. Results: Patients treated with P2Et were discharged on average after 7.4 days of admission vs. 9.6 days in the placebo group. Although a decrease in proinflammatory cytokines such as G-CSF, IL-15, IL-12, IL-6, IP10, MCP-1, MCP-2 and IL-18 was observed in both groups, P2Et decreased to a greater extent G-CSF, IL-6 and IL-18 among others, which are related to lower recovery of patients in the long term. The frequency of T lymphocytes (LT) CD3+, LT double negative (CD3+CD4-CD8-), NK cells increased in the P2Et group where the population of eosinophils was also significantly reduced. In the murine bleomycin model, P2Et also reduced lung inflammation and fibrosis. P2Et was able to reduce the viral replication of murine and human coronaviruses in vitro, showing its dual antiviral and anti-inflammatory role, key in disease control. Conclusions: Taken together these results suggest that P2Et could be consider as a good co-adjuvant in the treatment of COVID-19. Clinical trail registration: https://clinicaltrials.gov/ct2/show/NCT04410510, identifier: NCT04410510.

c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 innate lymphoid cells.

Group-2 innate lymphoid cells (ILC2s), which are involved in type 2 inflammatory diseases such as allergy, can exhibit immunological memory, but the basis of this ILC2 "trained immunity" has remained unclear. Here, we found that stimulation with IL-33/IL-25 or exposure to the allergen papain induces the expression of the transcription factor c-Maf in mouse ILC2s. Chronic papain exposure results in high production of IL-5 and IL-13 cytokines and lung eosinophil recruitment, effects that are blocked by c-Maf deletion in ILCs. Transcriptomic analysis revealed that knockdown of c-Maf in ILC2s suppresses expression of type 2 cytokine genes, as well as of genes linked to a memory-like phenotype. Consistently, c-Maf was found highly expressed in human adult ILC2s but absent in cord blood and required for cytokine production in isolated human ILC2s. Furthermore, c-Maf-deficient mouse or human ILC2s failed to exhibit strengthened ("trained") responses upon repeated challenge. Thus, the expression of c-Maf is indispensable for optimal type 2 cytokine production and proper memory-like responses in group-2 innate lymphoid cells.

PPARÉ£ drives IL-33-dependent ILC2 pro-tumoral functions.

Group 2 innate lymphoid cells (ILC2s) play a critical role in protection against helminths and in diverse inflammatory diseases by responding to soluble factors such as the alarmin IL-33, that is often overexpressed in cancer. Nonetheless, regulatory factors that dictate ILC2 functions remain poorly studied. Here, we show that peroxisome proliferator-activated receptor gamma (PPARγ) is selectively expressed in ILC2s in humans and in mice, acting as a central functional regulator. Pharmacologic inhibition or genetic deletion of PPARγ in ILC2s significantly impair IL-33-induced Type-2 cytokine production and mitochondrial fitness. Further, PPARγ blockade in ILC2s disrupts their pro-tumoral effect induced by IL-33-secreting cancer cells. Lastly, genetic ablation of PPARγ in ILC2s significantly suppresses tumor growth in vivo. Our findings highlight a crucial role for PPARγ in supporting the IL-33 dependent pro-tumorigenic role of ILC2s and suggest that PPARγ can be considered as a druggable pathway in ILC2s to inhibit their effector functions. Hence, PPARγ targeting might be exploited in cancer immunotherapy and in other ILC2-driven mediated disorders, such as asthma and allergy.

An Immunomodulatory Gallotanin-Rich Fraction From Caesalpinia spinosa Enhances the Therapeutic Effect of Anti-PD-L1 in Melanoma.

PD-1/PD-L1 pathway plays a role in inhibiting immune response. Therapeutic antibodies aimed at blocking the PD-1/PD-L1 interaction have entered clinical development and have been approved for a variety of cancers. However, the clinical benefits are reduced to a group of patients. The research in combined therapies, which allow for a greater response, is strongly encouraging. We previously characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) with antitumor activity in both melanoma and breast carcinoma, as well as immunomodulatory activity. We hypothesize that the combined treatment with P2Et and anti-PD-L1 can improve the antitumor response through an additive antitumor effect. We investigated the antitumor and immunomodulatory activity of P2Et and anti-PD-L1 combined therapy in B16-F10 melanoma and 4T1 breast carcinoma. We analyzed tumor growth, hematologic parameters, T cell counts, cytokine expression, and T cell cytotoxicity. In the melanoma model, combined P2Et and anti-PD-L1 therapy has the following effects: decrease in tumor size; increase in the number of activated CD4+ and CD8+ T cells; decrease in the number of suppressor myeloid cells; increase in PD-L1 expression; decrease in the frequency of CD8+ T cell expressing PD-1; improvement in the cytotoxic activity of T cells; and increase in the IFN γ secretion. In the breast cancer model, P2Et and PD-L1 alone or in combination show antitumor effect with no clear additive effect. This study shows that combined therapy of P2Et and anti-PD-L1 can improve antitumor response in a melanoma model by activating the immune response and neutralizing immunosuppressive mechanisms.

Immune system activation by natural products and complex fractions: a network pharmacology approach in cancer treatment.

Natural products and traditional herbal medicine are an important source of alternative bioactive compounds but very few plant-based preparations have been scientifically evaluated and validated for their potential as medical treatments. However, a promising field in the current therapies based on plant-derived compounds is the study of their immunomodulation properties and their capacity to activate the immune system to fight against multifactorial diseases like cancer. In this review we discuss how network pharmacology could help to characterize and validate natural single molecules or more complex preparations as promising cancer therapies based on their multitarget capacities.

Immunosuppressive Mediators Impair Proinflammatory Innate Lymphoid Cell Function in Human Malignant Melanoma.

Innate lymphoid cells (ILC) are a family of immune cells that are emerging as potent orchestrators of immune responses. In cancer, ILCs display both pro- and antitumorigenic functions depending on the nature of the tumor and the involved ILC subset. Little is known about the ILC-tumor cross-talk in human melanoma. Here, we showed that ILC1s were enriched but functionally impaired in cytokine secretion in both peripheral blood mononuclear cells and tumor-infiltrated lymph nodes of melanoma patients. These findings were confirmed in vivo in murine cutaneous melanoma. Multiple immunosuppressive mechanisms are described in the melanoma microenvironment. Among others, adenosine and kynurenines were shown to suppress antitumor immune responses. By exposing ILCs to adenosine and kynurenines, we observed a similar shift toward the ILC1 subset distribution and impairment in proinflammatory cytokine production to that of patient samples studied ex vivo. Thus, we hypothesized that the immunosuppressive microenvironment of malignant melanoma might shape ILC subpopulations. Hence, we provide a rationale for the use of drugs targeting adenosine and kynurenine pathways in melanoma patients.

Detecting and analyzing murine innate lymphoid cells.

During the last 10 years, different subsets of Innate lymphoid cells (ILCs) have been identified in murine models. ILCs play and important role in maintaining immune barriers, tissue homeostasis, and are able to regulate the immune response in several anatomic sites. They can be found in lymphoid and non-lymphoid organs of adult mice but are mainly tissue-resident cells that can expand locally under physiologic or pathologic conditions (Gasteiger, Fan, Dikiy, Lee, & Rudensky, 2015). Because ILCs need to be identified by a complex combination of several cell-surface and intracellular markers and by their production of specific sets of cytokines, multiparametric flow cytometry remains one of the most efficient methods to analyze and isolate the different ILC sub-populations. This chapter describes how ILCs can be identified in different murine organs and how ILC subsets can be isolated and functionally analyzed.

CD56 as a marker of an ILC1-like population with NK cell properties that is functionally impaired in AML.

An understanding of natural killer (NK) cell physiology in acute myeloid leukemia (AML) has led to the use of NK cell transfer in patients, demonstrating promising clinical results. However, AML is still characterized by a high relapse rate and poor overall survival. In addition to conventional NKs that can be considered the innate counterparts of CD8 T cells, another family of innate lymphocytes has been recently described with phenotypes and functions mirroring those of helper CD4 T cells. Here, in blood and tissues, we identified a CD56+ innate cell population harboring mixed transcriptional and phenotypic attributes of conventional helper innate lymphoid cells (ILCs) and lytic NK cells. These CD56+ ILC1-like cells possess strong cytotoxic capacities that are impaired in AML patients at diagnosis but are restored upon remission. Their cytotoxicity is KIR independent and relies on the expression of TRAIL, NKp30, NKp80, and NKG2A. However, the presence of leukemic blasts, HLA-E-positive cells, and/or transforming growth factor-ß1 (TGF-ß1) strongly affect their cytotoxic potential, at least partially by reducing the expression of cytotoxic-related molecules. Notably, CD56+ ILC1-like cells are also present in the NK cell preparations used in NK transfer-based clinical trials. Overall, we identified an NK cell-related CD56+ ILC population involved in tumor immunosurveillance in humans, and we propose that restoring their functions with anti-NKG2A antibodies and/or small molecules inhibiting TGF-ß1 might represent a novel strategy for improving current immunotherapies.

Immunophenotyping of Human Innate Lymphoid Cells.

In the last years, the family of innate lymphocytes has been growing following the discovery of innate lymphoid cells (ILCs). ILCs are lymphocytes able to rapidly produce a wide range of soluble mediators in an antigen-independent fashion. So far, three main subsets of ILCs have been discovered, ILC1, ILC2, and ILC3, expressing respectively the transcription factors T-bet, GATA3, and Rorγt and secreting distinct types of cytokines. After their discovery, several studies showed that different pathologies, such as allergic airway diseases and inflammatory disorders, are sustained by dysfunctional ILCs before adaptive immune sets in. In this regard, considerable efforts are currently performed to harmonize the identification and monitoring of ILCs in healthy and pathologic conditions to streamline a uniform immunophenotyping. Standardized ILC monitoring techniques will accelerate our understanding of these effector innate immune cells and ultimately facilitate their targeting in the context of infection, cancer, autoimmune disease, and transplantation.

Peripheral Innate Lymphoid Cells Are Increased in First Line Metastatic Colorectal Carcinoma Patients: A Negative Correlation With Th1 Immune Responses.

Several distinct innate lymphoid cell (ILC) populations have been recently identified and shown to play a critical role in the immediate immune defense. In the context of tumors, there is evidence to support a dual role for ILCs with pro- or antitumor effects, depending on the ILC subset and the type of cancer. This ambivalent role has been particularly well-described in colorectal cancer models (CRC), but the presence and the evolution of ILCs in the peripheral blood of metastatic CRC (mCRC) patients have not yet been explored. Here, we investigated the distribution of ILC subsets in 96 mCRC patients who were prospectively included in the "Epitopes-CRC02" trial. Peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry at metastatic diagnosis and after 3-months of treatment. The treatments consisted of Oxaliplatin-based chemotherapies for 76% of the patients or Folfiri (5FU, Irinotecan) chemotherapies for 14% of patients. Compared to healthy donors, the frequency of total ILCs was dramatically increased at metastatic diagnosis. CD56+ ILC1-like cells were expanded, whereas ILC2, NCR- ILCP and NCR+ ILCP subsets were decreased. Combined analysis with the systemic anti-telomerase hTERT Th1 CD4 response revealed that patients with low anti-TERT Th1 CD4 responses had the highest frequencies of total ILCs at diagnosis. Of those, 91% had synchronous metastases, and their median progression-free survival was 7.43 months (vs. 9.17 months for the other patients). In these patients, ILC1 and ILC2 were significantly decreased, whereas CD56+ ILC1-like cells were significantly increased compared to patients with low frequency of total ILCs and high anti-TERT responses. After treatment, the NCR+ ILCP were further decreased irrespective of the chemotherapy regimen, whereas the balance between ILC1 and CD56+ ILC1-like cells was modulated mainly by the Folfiri regimen in favor of ILC1. Altogether our results describe the effects of different chemotherapies on ILCs in mCRC patients. We also establish for the first time a link between frequency of ILCs and anti-tumor CD4 T cell responses in cancer patients. Thus, our study supports an interest in monitoring ILCs during cancer therapy to possibly identify predictive biomarkers in mCRC.

Prophylactic vs. Therapeutic Treatment With P2Et Polyphenol-Rich Extract Has Opposite Effects on Tumor Growth.

Polyphenols have tumoricidal effects via anti-proliferative, anti-angiogenic and cytotoxic mechanisms and have recently been demonstrated to modulate the immune response through their anti- or pro- oxidant activity. Nevertheless, it remains controversial whether antioxidant-rich supplements have real beneficial effects on health, especially in complex diseases such as cancer. We previously identified a polyphenol-rich extract obtained from Caesalpinia spinosa (P2Et) with anti-tumor activity in both breast carcinoma and melanoma. The present work evaluated the ability of P2Et extract to modulate the immune system in either the steady state or following tumor challenge. We found that the prophylactic treatment of healthy mice increased the number of CD4+ and CD8+ activated T, NK, regulatory T, dendritic and myeloid-derived suppressor cells in lymphoid organs together with a significant increase in plasma IL-6. Interestingly, this pre-conditioning of the host immune system with P2Et did not involve a protective effect against the control of tumor growth and metastasis in transplantable models of melanoma (B16) and breast cancer (4T1), but in contrast, a detrimental effect was observed in both models. We further demonstrated that this effect was at least partly due to an increase in regulatory T cells, myeloid-derived suppressor cells, and proinflammatory cytokines, with a concomitant decrease in CD4+ and CD8+ T cells. Taken together, these results suggest that the anti-tumor and immunomodulation properties of the P2Et extract critically depend on the presence of the tumor and might be mediated by the complex interactions between the tumor cells and the other components of the tumor microenvironment.

Human innate lymphoid cells (ILCs): Toward a uniform immune-phenotyping.

Helper innate lymphoid cells (ILCs), the most recently identified population of the ILC family, play a fundamental role in the restoration of tissue integrity, in the protection against infiltrating pathogens as well as in tumor immune-surveillance. ILCs have been divided into three main subsets, ILC1, ILC2, and ILC3, that can be specifically activated by different signals coming either indirectly from pathogens or from other cell populations, including cancer cells. Following activation, ILCs are in turn able to promptly secrete a wide range of soluble mediators that modulate effector cell functions. The discovery and the study of these immune cells is now offering important opportunities for innovative therapies of allergic airway diseases, inflammatory disorders and might be crucial for the discovery of new targets for the therapy of cancer. It is therefore fundamental that the scientific community establishes harmonized guidelines to obtain a consensus in the identification and phenotypical and functional characterization of ILCs. © 2018 International Clinical Cytometry Society.

Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis.

Group 2 innate lymphoid cells (ILC2s) are involved in human diseases, such as allergy, atopic dermatitis and nasal polyposis, but their function in human cancer remains unclear. Here we show that, in acute promyelocytic leukaemia (APL), ILC2s are increased and hyper-activated through the interaction of CRTH2 and NKp30 with elevated tumour-derived PGD2 and B7H6, respectively. ILC2s, in turn, activate monocytic myeloid-derived suppressor cells (M-MDSCs) via IL-13 secretion. Upon treating APL with all-trans retinoic acid and achieving complete remission, the levels of PGD2, NKp30, ILC2s, IL-13 and M-MDSCs are restored. Similarly, disruption of this tumour immunosuppressive axis by specifically blocking PGD2, IL-13 and NKp30 partially restores ILC2 and M-MDSC levels and results in increased survival. Thus, using APL as a model, we uncover a tolerogenic pathway that may represent a relevant immunosuppressive, therapeutic targetable, mechanism operating in various human tumour types, as supported by our observations in prostate cancer.Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth.

Standardized Extract from Caesalpinia spinosa is Cytotoxic Over Cancer Stem Cells and Enhance Anticancer Activity of Doxorubicin.

Cancer stem cells (CSC) are the primary cell type responsible for metastasis and relapse. ABC-transporters are integral membrane proteins involved in the translocation of substrates across membranes protecting CSC from chemotherapeutic agents. A plant extract derived from C. spinosa (P2Et) previously investigated for its antitumor activity has been shown to reduce lung and spleen metastasis in mice that have been transplanted with breast cancer cells, suggesting that P2Et has a significant activity against cancer stem cells (CSC). P2Et extract was thoroughly characterized by HPLC/MS. The cytotoxicity of P2Et extract was evaluated using a MTT assay in human and murine cell lines with different profiles of resistance, by Pgp overexpression or by enrichment in cancer stem cells. The synergistic effect of P2Et with doxorubicin was evaluated in vitro in several cell lines and in vivo in mice transplanted with TS/A cells, a highly resistant cell line and enriched in CD44[Formula: see text]CD24[Formula: see text]CSC. The chromatographic fingerprint of P2Et extract revealed 13 gallotannins. We also found that P2Et extract was cytotoxic to cells regardless of their resistant phenotype. Similarly, complementary activities were observed as drug efflux reversion and antioxidant activity. Short-treatment with P2Et extract, revealed a synergistic effect with doxorubicin in resistant cell lines. In vivo the P2Et increases mice survival in a TS/A breast cancer model associated with augmentation of calreticulin expression. Our results suggest that P2Et treatment could be used as adjuvant along with conventional chemotherapy to treat tumors with a MDR phenotype or with high frequency of CSC.