Plasticity of High-Density Neutrophils in Multiple Myeloma is Associated with Increased Autophagy via STAT3

Abstract

In both monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM) patients, immune functions are variably impaired, and there is a high risk of bacterial infections. Neutrophils are the most abundant circulating leukocytes and constitute the first line of host defense. Since little is known about the contribution of autophagy in the neutrophil function of MGUS and MM patients, we investigated the basal autophagy flux in freshly sorted neutrophils of patients and tested the plastic response of healthy neutrophils to soluble factors of MM. In freshly sorted high-density neutrophils obtained from patients with MGUS and MM or healthy subjects, we found a progressive autophagy trigger associated with soluble factors circulating in both peripheral blood and bone marrow, associated with increased IFNγ and pSTAT3S727. In normal high-density neutrophils, the formation of acidic vesicular organelles, a morphological characteristic of autophagy, could be induced after exposure for three hours with myeloma conditioned media or MM sera, an effect associated with increased phosphorylation of STAT3-pS727 and reverted by treatment with pan-JAK2 inhibitor ruxolitinib. Taken together, our data suggest that soluble factors in MM can trigger contemporary JAK2 signaling and autophagy in neutrophils, targetable with ruxolitinib.

High-density neutrophils in MGUS and multiple myeloma are dysfunctional and immune-suppressive due to increased STAT3 downstream signaling

“…In this work, we investigated immune-phenotype and function of mature, high-density neutrophils circulating in peripheral blood of MM and MGUS patients, aiming that HDNs could have a role in the complex pathogenesis of myeloma development and mirror the interplay between the tumor growth in the bone marrow and the myeloid development. To exclude the contamination with eosinophils or monocytes, we worked with highly-purified freshly-isolated HDNs 38,54 .…”

“…There is an emerging interest in identifying the molecular machinery involved in the IFN response in the MM microenvironment, since several cellular types show an IFNAR1 related signature in response to type 1 interferon secretion by myeloma cells 72 , while type 3 interferon is reduced 73 due to anergic NK and T-cells 61 also in the asymptomatic MGUS phase 74 . Further effort in our lab is currently focused to identify the source of extracellular IFN-γ in the paracrine loop sustaining MM expansion, and we have evidence of out-of-lineage, low-rate synthesis of IFN-γ in MM-HDNs 38 .…”

“…Both LDNs and HDNs promote tumor growth and chemo-refractoriness, contributing to the MM pathogenesis in mice 33,35,36 . In humans, LDNs, firstly reported in chronic inflammatory disease, induce in cancer T-cell anergy via secretion of aminoacid degrading enzymes arginase and tryptophan 37,38 .…”

“…We found ARG1 gene upregulation among the up-regulated genes in MM-HDNs compared to healthy HDNs. Since our previous work showed that ARG1, a transcriptional target of STAT-3 47,48 , STAT-5 49 and STAT-6 50,51 , is increased in granulocytic-like myeloid derived suppressor cells in MM 28,38,52 , associated to inferior outcome after bortezomib treatment 28 , we explored its expression in both MGUS-and MM-HDNs.…”

“…After washing, cells were further immune-magnetically sorted using the EasySep human neutrophil Isolation kit (StemCell Technology, cat #17957) 54 . HDN purity, maturity and viability were checked by morphology, as previously described 38 and flow cytometry as CD45 + CD11b + CD15 + CD14cells ( Supplementary Fig. 5).…”