Supplementary Material for: Isoliquiritigenin Ameliorates Indomethacin-Induced Small Intestinal Damage by Inhibiting NOD-Like Receptor Family, Pyrin Domain-Containing 3 Inflammasome Activation

Activation of the NOD-Like Receptor Family, Pyrin Domain-Containing 3 (NLRP3) inflammasome, which consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1, triggers pro-caspase-1 cleavage promoting the processing of pro-interleukin (IL)-1β into mature IL-1β, which is critical for the development of non-steroidal anti-inflammatory drug (NSAID)-induced enteropathy. We investigated the effects of isoliquiritigenin, a flavonoid derived from the roots of <i>Glycyrrhiza</i> species, on NSAID-induced small intestinal damage and the inflammasome activation. To induce enteropathy, mice were administered indomethacin by gavage with or without isoliquiritigenin pretreatment. Some mice received an intraperitoneal injection of recombinant murine IL-1β in addition to isoliquiritigenin and indomethacin. Indomethacin induced small intestinal damage and increased protein levels of cleaved caspase-1 and mature IL-1β in the small intestine. Treatment with 7.5 and 75 mg/kg isoliquiritigenin inhibited indomethacin-induced small intestinal damage by 40 and 56%, respectively. Isoliquiritigenin also inhibited the indomethacin-induced increase in cleaved caspase-1 and mature IL-1β protein levels, whereas it did not affect the mRNA expression of NLRP3, ASC, caspase-1, and IL-1β. Protection against intestinal damage in isoliquiritigenin-treated mice was completely abolished with exogenous IL-1β. NLRP3^–/– and caspase-1^–/– mice exhibited resistance to intestinal damage, and isoliquiritigenin treatment failed to inhibit the damage in NLRP3^–/– and caspase-1^–/– mice. Isoliquiritigenin prevents NSAID-induced small intestinal damage by inhibiting NLRP3 inflammasome activation

Supplementary Material for: Potential Role of Gr-1⁺ CD8⁺ T Lymphocytes as a Source of Interferon-γ and M1/M2 Polarization during the Acute Phase of Murine <b><i>Legionella pneumophila</i></b> Pneumonia

In this study, we analyzed interferon (IFN)-γ-producing cells and M1/M2 macrophage polarization in <i>Legionella pneumophila</i> pneumonia following anti-Gr-1 antibody treatment. Anti-Gr-1 treatment induced an M1-to-M2 shift of macrophage subtypes in the lungs and weakly in the peripheral blood, which was associated with increased mortality in legionella-infected mice. CD8⁺ T lymphocytes and natural killer cells were the dominant sources of IFN-γ in the acute phase, and anti-Gr-1 treatment reduced the number of IFN-γ-producing CD8⁺ T lymphocytes. In the CD3-gated population, most Gr-1-positive cells were CD8⁺ T lymphocytes in the lungs and lymph nodes (LNs) of infected mice. Additionally, the number of IFN-γ-producing Gr-1⁺ CD8⁺ T lymphocytes in the lungs and LNs increased 2 and 4 days after <i>L. pneumophila</i> infection, with anti-Gr-1 treatment attenuating these populations. Antibody staining revealed that Gr-1⁺ CD8⁺ T lymphocytes were Ly6C-positive cells rather than Ly6G, a phenotype regarded as memory type cells. Furthermore, the adoptive transfer of Gr-1⁺ CD8⁺ T lymphocytes induced increases in IFN-γ, M1 shifting and reduced bacterial number in the <i>Legionella</i> pneumonia model. These data identified Ly6C⁺ CD8⁺ T lymphocytes as a source of IFN-γ in innate immunity and partially associated with reduced IFN-γ production, M2 polarization, and high mortality in anti-Gr-1 antibody-treated mice with <i>L. pneumophila</i> pneumonia

Supplementary Material for: Contemplation of the effect of nivolumab plus cabosantinib therapy on cerebral hemorrhage in patients with brain metastasis of renal cell carcinoma: A case report

Although the response to combination therapy has been reported in patients with brain metastases from advanced renal cancer, treatment-related cerebral hemorrhage has not been adequately studied. The CheckMate 9ER clinical trial of nivolumab and cabozantinib excluded patients with brain metastases. Therefore, the associated treatment outcomes in these patients with brain metastases are unclear. Herein, we report a case of bleeding from brain metastases in a patient with advanced renal cancer after gamma knife combination therapy with nivolumab and cabozantinib. Fortunately, the cerebral hemorrhage of the patient was alleviated by conservative treatment. Despite treatment interruption, the metastatic lesions reduced in size, and treatment was gradually resumed. In this case study, we report the risk of cerebral hemorrhage in combination therapy for brain metastasis cases, how to manage hemorrhage cases, and their prognosis