RAC2 gain-of-function variants causing inborn error of immunity drive NLRP3 inflammasome activation.

A growing number of patients presenting severe combined immunodeficiencies attributed to monoallelic RAC2 variants have been identified. The expression of the RHO GTPase RAC2 is restricted to the hematopoietic lineage. RAC2 variants have been described to cause immunodeficiencies associated with high frequency of infection, leukopenia, and autoinflammatory features. Here, we show that specific RAC2 activating mutations induce the NLRP3 inflammasome activation leading to the secretion of IL-1ß and IL-18 from macrophages. This activation depends on the activation state of the RAC2 variant and is mediated by the downstream kinase PAK1. Inhibiting the RAC2-PAK1-NLRP3 inflammasome pathway might be considered as a potential treatment for these patients.

The generation, activation, and polarization of monocyte-derived macrophages in human malignancies.

Macrophages are immune cells that originate from embryogenesis or from the differentiation of monocytes. They can adopt numerous phenotypes depending on their origin, tissue distribution and in response to different stimuli and tissue environment. Thus, in vivo, macrophages are endowed with a continuum of phenotypes that are rarely strictly pro-inflammatory or anti-inflammatory and exhibit a broad expression profile that sweeps over the whole polarization spectrum. Schematically, three main macrophage subpopulations coexist in human tissues: naïve macrophages also called M0, pro-inflammatory macrophages referred as M1 macrophages, and anti-inflammatory macrophages also known as M2 macrophages. Naïve macrophages display phagocytic functions, recognize pathogenic agents, and rapidly undergo polarization towards pro or anti-inflammatory macrophages to acquire their full panel of functions. Pro-inflammatory macrophages are widely involved in inflammatory response, during which they exert anti-microbial and anti-tumoral functions. By contrast, anti-inflammatory macrophages are implicated in the resolution of inflammation, the phagocytosis of cell debris and tissue reparation following injuries. Macrophages also play important deleterious or beneficial roles in the initiation and progression of different pathophysiological settings including solid and hematopoietic cancers. A better understanding of the molecular mechanisms involved in the generation, activation and polarization of macrophages is a prerequisite for the development of new therapeutic strategies to modulate macrophages functions in pathological situations.

Reprogramming monocyte-derived macrophages through caspase inhibition.

Macrophages are widely distributed innate immune cells that play an indispensable role in a variety of physiologic and pathologic processes, including organ development, host defense, acute and chronic inflammation, solid and hematopoietic cancers. Beyond their inextricable role as conveyors of programmed cell death, we have previously highlighted that caspases exert non-apoptotic functions, especially during the differentiation of monocyte-derived cells in response to CSF-1. Here, we found that non-canonic cleavages of caspases, reflecting their activation, are maintained during IL-4-induced monocyte-derived macrophages polarization. Moreover, Emricasan, a pan-caspase inhibitor that demonstrated promising preclinical activity in various diseases and safely entered clinical testing for the treatment of liver failure, prevents the generation and the anti-inflammatory polarization of monocyte-derived macrophages ex vivo. Interestingly, caspase inhibition also triggered the reprogramming of monocyte-derived cells evidenced by RNA sequencing. Taken together, our findings position Emricasan as a potential alternative to current therapies for reprogramming macrophages in diseases driven by monocyte-derived macrophages.

P2RY2-AKT activation is a therapeutically actionable consequence of XPO1 inhibition in acute myeloid leukemia.

Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice.

Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.

Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression.

Background: Cathepsin L (Ctsl) is a cysteine protease mainly located within the endosomal/lysosomal cell compartment. High expression of Ctsl indicates poor prognosis in human breast cancer. However, the cell type-specific Ctsl functions responsible for this association remain elusive. Methods: Because constitutive Ctsl-/- mice develop a complex phenotype, we developed a conditional model allowing for cell type-specific inactivation of Ctsl in mammary epithelium or myeloid cells in the transgenic mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer model. Results: Ctsl ablation in mammary epithelial cells resulted in delayed initiation and end-stage of cancers. The latter displayed large dead cell areas. Inducible in vitro deletion of Ctsl in MMTV-PyMT-derived breast cancer cells revealed expansion of the acidic cell compartment, alteration of intracellular amino acid levels, and impaired mTOR signaling. In consequence, Ctsl-deficient cells exhibited slow growth rates and high apoptosis susceptibility. In contrast to Ctsl-deficient mammary epithelium, selective knockout of Ctsl in myeloid cells had no effects on primary tumors, but promoted lung metastasis formation. Conclusions: Our cell type-specific in vivo analysis provides strong evidence for a cancer cell-intrinsic, tumor-promoting role of Ctsl in primary breast cancer, whereas metastasis is negatively regulated by Ctsl expressed by bone marrow-derived cells.

IL-34 and CSF-1 display an equivalent macrophage differentiation ability but a different polarization potential.

CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages.