Granulocyte Derived Resistin Induces Classical Monocyte Repartition and Immune Suppression in CMML

Introduction:

Chronic myelomonocytic leukemia (CMML) is a clonal malignancy characterized by overlapping myeloid dysplasia and proliferation with persisting monocytosis. Characteristic repartitioning of classical monocytes (CD14⁺/CD16^-/low) is now a supporting diagnostic criterion (WHO5), though its mechanistic basis remains unknown. While monocytes are the cardinal malignant cell type in CMML, as a stem cell neoplasm the disease clone comprises most lineages and differentiation stages. Granulocytes are the most abundant circulating blood cells and play key roles in immune response via cytokine and growth factor secretion. They too are typically expanded, dysfunctional and linked to poor prognosis in CMML; their pathogenic contribution however remains understudied. We hypothesized that granulocytes play a critical role in CMML maintenance and progression.

Methods:

We isolated granulocytes via negative selection from healthy (n=19) and CMML (n=26) peripheral blood for immunophenotype, transcriptome and functional analyses. Phagocytic capacity was determined using zymosal uptake assays. Resistin plasma levels were determined by ELISA. Resistin’s role in CMML biology was studied by RNA-seq, flow cytometry and a series of cell viability, monocyte differentiation and T cell profiling assays. Clinical correlates, including survival analyses, were determined from linked patient metadata.

Results:

Compared with healthy age-matched controls, CMML granulocytes exhibited defective maturation with reduced granularity, decreased mature CD11b+/CD16+ fraction and higher CD66 expression. Immature status was independent of clinical features and mutation profile. CMML granulocytes were also defective in phagocytic capacity compared to healthy controls. RNA-seq clearly distinguished between granulocytes from healthy (n=14) and CMML (n=14) patients. Upregulated genes in CMML are involved in pathways linked to proliferation and Myc activity, with defensins and neutrophil elastase (ELANE) among the top 20 upregulated genes. Notably, RETN, which encodes the cysteine-rich peptide hormone and inflammatory mediator resistin, was upregulated 100-fold in CMML granulocytes; but not differentially expressed in CMML bulk MNCs, sorted monocytes, or stem cells compared to healthy counterparts. Accordingly, resistin protein levels were 10-fold higher in plasma from CMML patients compared with age-matched controls; levels positively correlated with granulocyte RETN RNA expression, validating CMML granulocytes as the primary source of raised resistin levels. Patients with high secreted resistin levels also displayed worse overall survival.

Remarkably, exposure of healthy monocytes to exogenous recombinant resistin induced classical monocyte repartition, recapitulating the pattern seen in CMML. This phenotype was confirmed to be mediated via resistin’s interaction with TLR4. Healthy monocyte differentiation to macrophages was also suppressed in the presence of recombinant resistin. We next performed RNA-seq on resistin- and LPS-treated healthy monocytes, revealing gene expression patterns consistent with resistin-induced maintenance of a classical monocyte state. Genes induced by resistin are involved in immune suppression and myeloid-derived suppressor phenotype. We then overlapped these with genes differentially expressed in CMML monocytes, revealing candidate in vivo targets of resistin. These included SEMA4A, SDC2, SPP1 and VIM: genes involved in M2 polarization and immune suppression. We confirmed SEMA4A induction at the protein level upon resistin treatment, and observed higher SEMA4A levels linked to poor prognosis and an immune suppressive cellular phenotype. Consistent with known roles for SEMA4A in Treg expansion and Th2/Th1 ratio skewing, CMML patients displayed higher T-regs and Th2/Th1 ratio compared with healthy controls, apparently corresponding with associated resistin levels.

Conclusions:

We demonstrate that CMML granulocytes are functionally immature and produce high levels of resistin, directly contributing to the classical monocytosis phenotype. High resistin plasma levels correlate with expanded Tregs and induce immune suppressive features in CMML monocytes via downstream mediators including SEMA4A. Thus, approaches targeting resistin or blocking the resistin-TLR4 interaction may have therapeutic value in CMML.