Tumor-associated inflammation leads to dysregulated cytokine production that promotes tumor immune evasion and anti-tumor immunity dysfunction. In advanced stage breast cancer, the proinflammatory cytokine IL-1β is overexpressed due to large proportions of activated myeloid cells in the tumor microenvironment (TME). Here, we demonstrate the role of the host nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing 3 (NLRP3) inflammasome in metastatic breast cancer. In vitro, we show that stimulation of THP-1 cells with conditioned media collected from MDA-MB-468 cells induced NLRP3 activation and increased Pdcd1l1 expression. In vivo, mice deficient in NLRP3 orthotopically implanted with metastatic breast cancer cell line (E0771) showed significant reduction in tumor growth (p < 0.05) and increased survival (p < 0.01). Inhibition of NLRP3 with the small molecule OLT1177® reduced expression of Pdcd1l1 (p < 0.001), Casp1 (p < 0.01) and Il1b (p < 0.01) in primary tumors. Furthermore, tumor-bearing mice receiving OLT1177® showed reduced infiltration of myeloid-derived suppressor cells (MDSCs) (p < 0.001) and increased CD8+ T cells (p < 0.05) and NK cells (p < 0.05) in the TME. NLRP3 inhibition in addition to anti-PD-1 treatment significantly reduced tumor growth from the monotherapies (p < 0.05). These data define NLRP3 activation as a key driver of immune suppression in metastatic breast cancers. Furthermore, this study suggests NLRP3 as a valid target to increase efficacy of immunotherapy with checkpoint inhibitor in metastatic breast cancers.

The IL-1 Family member IL-38 has been characterized as an anti-inflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL 38 in the small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL 38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL 38 deficient mice develop more slowly. This reduction in size is likely due to downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, Olfm4) expression compared with wild type organoids. IL-38 binding to IL-1R6 is postulated to recruit the co-receptor IL-1R9. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL 1R9 deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL 38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL 38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild type mice, IL-38 stimulation induced low concentrations of IL-1β which contribute to organoid growth. However, high concentrations of IL 1beta have detrimental effects on the cultures that were prevented by treatment with recombinant IL 38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an anti-inflammatory molecule in the SI, maintaining homeostasis.

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