Several studies have documented the ability of flavonoids to sensitize cancer cells to chemotherapeutics and reverse multidrug resistance by inhibition of efflux pumps (adenosine triphosphate‐binding cassette transporters), apoptosis activation, and cell cycle arrest. In this study, the flavonoid rutin (quercetin 3‐O‐β‐d‐rutinoside) was investigated as chemosensitizer towards two different human epithelial breast cancer cell lines: (i) MB‐MDA‐231, selected as representative for triple‐negative breast cancer and (ii) MCF‐7 used as a well‐characterized model of HER2‐negative breast cancer. To assess the cytocompatibility of rutin against non‐cancer cells, primary human mammary fibroblasts were used as control and non‐target cells. In MDA‐MB‐231 cells, 20 μM rutin enhanced cytotoxicity related to cyclophosphamide and methotrexate. Rutin significantly (p < 0.05) increased the anticancer activity of both chemotherapeutics, at 24–48–72 h, and decreased the activity of the adenosine triphosphate‐binding cassette transporters, namely, P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP). Flow cytometry analysis showed 20 μM and 50 μM rutin arrested cell cycle at G2/M and G0/G1 phases, respectively, significantly promoting cell apoptosis. Rutin, via non‐selective inhibition of P‐gp and BCRP pumps, efficiently reverses multidrug resistance and restores chemosensitivity to cyclophosphamide and cyclophosphamide of human chemoresistant, triple‐negative breast cancer cells, successfully arresting cell cycle progression. Copyright © 2017 John Wiley & Sons, Ltd.

Therapies counteracting pathogen-induced immune modulation of the host responses represent a promising improvement for tuberculosis treatments. Despite the documented role of host immune heterogeneity and bacterial genotypic background in determining infection outcomes, their interplay remains largely uncharacterized. We investigated the Mycobacterium tuberculosis (MTB)-human macrophage interaction, considering both macrophage phenotypic variability and MTB genetic diversity. Using single-cell techniques, we show how diverse MTB lineages fine-tune phagosome acidification differently based on macrophage phenotype (M1, M2), revealing very heterogeneous host-pathogen interactions. Our findings underscore the multiplicity of outcomes due to the combinatorial interplay between MTB lineages and macrophage phenotypes, which may have implications for the proper design of host-directed therapies. IMPORTANCE: The intricate interplay among host, pathogen, and environmental factors significantly contributes to susceptibility and clinical presentation of tuberculosis. These multifaceted mechanisms are often overlooked in tuberculosis studies, hindering our comprehensive understanding of infection progression and impeding the development of effective host-directed therapies, which offer benefits such as reduced drug resistance emergence and heightened host compatibility. Contrary to the prevailing hypothesis, the results observed in this study demonstrate the nuanced response of different M. tuberculosis lineages within distinct macrophage phenotypes in terms of phagosome acidification. The findings of our investigation delineate a crucial yet under-characterized aspect of tuberculosis pathogenesis, emphasizing how the interactions between different M. tuberculosis lineages and macrophage phenotypes could significantly influence the efficacy of host-directed therapies, particularly those targeting phagolysosomal maturation. More broadly, recognizing the impact of both M. tuberculosis and macrophage heterogeneity is paramount in the development of effective strategies to combat tuberculosis.

The WHO mutation catalog is the reference for genotypic antimicrobial susceptibility predictions (ASPs) for Mycobacterium tuberculosis . To increase sensitivity, WHO endorsed additional grading rules to classify mutations with insufficient experimental evidence. For rifampin, this includes any non-silent rpoB mutation in the rifampin resistance-determining region (RRDR). For delamanid and pretomanid, the rules include loss-of-function (LoF) variants in six resistance genes, including fbiC . Here, we describe three edge cases in which these additional grading rules overcalled resistance. The first case was a low-frequency rpoB variant in RRDR caused by a sequencing artifact. The second case involved a tandem repeat region starting at the end of fbiC . Because the H37Rv reference genome carries 2.7 repeats, deletion of one or two full repeats caused mapping errors, making these deletions appear as minority variants that trigger the fbiC LoF rule. However, assembly showed that these deletions did not affect the fbiC coding region because the remaining repeat substituted for the deleted repeats. The third case was a fbiC frameshift in lineage 4.6.1 Uganda genotype strains, caused by a 19 bp duplication upstream of the same tandem repeat region. Although this frameshift affects the fbiC coding region and meets the fbiC LoF rule, sequence assembly revealed only a modest impact on the end of fbiC . Although catalog performance remains high, WHO and others have acknowledged that, in specific situations, exceptions to its additional grading rules may result in unintended overcalling of resistance. Thus, systematic external quality control and communication of edge cases are essential. IMPORTANCE: Antimicrobial susceptibility predictions (ASPs) for Mycobacterium tuberculosis using next-generation sequencing and the WHO mutation catalog are increasingly used for clinical decision-making. This avoids systematic false-resistant results of real-time PCR- or hybridization-based ASP assays caused by mutations that do not confer resistance (e.g., synonymous rifampin resistance-determining region [RRDR] mutations) and, potentially, subsequent inappropriate treatment adjustments. However, we demonstrate that false-resistant results occur even using the WHO mutation catalog for rifampin, delamanid, and pretomanid. Similar exceptions linked to the additional grading rules of the catalog are possible for other drugs, underlining the importance of monitoring the performance of the catalog continuously.

Therapies counteracting pathogen-induced immune modulation of the host responses represent a promising improvement for tuberculosis treatments. Despite the documented role of host immune heterogeneity and bacterial genotypic background in determining infection outcomes, their interplay remains largely uncharacterized. We investigate the Mycobacterium tuberculosis (MTB)-human macrophage interaction considering both macrophage phenotypic variability and MTB genetic diversity. Using single-cell techniques, we show how diverse MTB lineages fine-tune phagosome acidification differently based on macrophage phenotype (M1, M2), revealing very heterogeneous host-pathogen interactions. Our findings underscore the multiplicity of outcomes due to the combinatorial interplay between MTB lineages and macrophage phenotypes, which may have implications in proper design for host-directed therapies. IMPORTANCE: The intricate interplay among host, pathogen, and environmental factors significantly contributes to susceptibility and clinical presentation of tuberculosis. These multifaceted mechanisms are often overlooked in tuberculosis studies, hindering our comprehensive understanding of infection progression and impeding the development of effective host-directed therapies which offer benefits such as reduced drug resistance emergence and heightened host compatibility. Contrary to the prevailing hypothesis, the results observed in this study demonstrate the nuanced response of different M. tuberculosis lineages within distinct macrophage phenotypes in terms of phagosome acidification. The findings of our investigation delineate a crucial yet under-characterized aspect of tuberculosis pathogenesis, emphasizing how the interactions between different M. tuberculosis lineages and macrophage phenotypes could significantly influence the efficacy of host-directed therapies, particularly those targeting phagolysosomal maturation. More broadly, recognizing the impact of both M. tuberculosis and macrophage heterogeneity is paramount in the development of effective strategies to combat tuberculosis.