Infiltration Of Immature Myeloid Cells Research Articles

Gut microbiota-mediated activation of GSDMD ignites colorectal tumorigenesis

Activation of Gasdermin D (GSDMD) results in its cleavage, oligomerization, and subsequent formation of plasma membrane pores, leading to a form of inflammatory cell death denoted as pyroptosis. The roles of GSDMD in inflammation and immune responses to infection are well documented. However, whether GSDMD also plays a role in sporadic cancer development, especially that in the gut epithelium, remains unknown. Here, we show that GSDMD is activated in colorectal tumors of both human and mouse origins. Ablation of GSDMD in a mouse model of sporadic colorectal cancer resulted in reduced tumor formation in the colon and rectum, suggesting a tumor-promoting role of the protein in the gut. Both antibiotic-mediated depletion of gut microbiota and pharmacological inhibition of NLRP3 inflammasome reduced the activation of GSDMD. Loss of GSDMD resulted in reduced infiltration of immature myeloid cells, and increased numbers of macrophages in colorectal tumors. Activation of GSDMD is also accompanied by the aggregation of the endosomal sorting complex required for transport (ESCRT) membrane repair proteins on the membrane of colorectal tumor cells, suggesting that active membrane repairment may prevent pyroptosis induced by the formation of GSDMD pore in tumor cells. Our results show that gut microbiota/NLRP3-mediated activation of GSDMD promotes the development of colorectal tumors, and supports the use of NLRP3 inhibitors to treat colon cancer.

Development of a Fully Immunocompetent Adoptive Transfer In Vivo Model of Npm1 cA;Idh2 R140Q;Flt3 ITD AML

AML is a highly heterogeneous disease, both genetically and phenotypically. This includes both intra- and inter-patient heterogeneity that significantly impacts prognosis and treatment responses. Mouse models are a powerful tool to further our understanding of fundamental disease biology and to advance novel therapies. While human cell line (CDX) and primary patient (PDX) xenografts are reproducible in vivo models, these models are by necessity immunodeficient. Transgenic mouse models provide an opportunity to study disease development driven by human relevant mutations within a fully immunocompetent system. However, drug development studies often require large numbers of mice and predictable time of disease development that is infeasible using primary transgenic animals. We therefore sought to develop a transgenic mouse model representative of human disease that was able to consistently be adoptively transferred into fully immunocompetent recipients. Our laboratory has interest in multiple targeted therapeutics and chose to focus on a model that may have sensitivity to multiple translationally relevant targeted drugs. We crossed the previously established Npm1 cA (Vassiliou et al 2011), Flt3 ITD (Lee et al 2007) and Idh2 R140Q (Shih et al 2017) Cre-inducible alleles to generate a triple mutant AML model ( Npm1 cA;Idh2 R140Q;Flt3 ITD). This model develops an aggressive AML with a median survival of 44 days. Complete blood counts at endpoint demonstrate significantly elevated white cells as well as profound anemia and thrombocytopenia. Npm1 cA;Idh2 R140Q;Flt3 ITD mice also have splenomegaly and infiltration of immature myeloid cells into both lymphoid and non-lymphoid tissues. Flow cytometry analysis of the blood, spleen and bone marrow shows a significant expansion of CD11b+cKit+ cells characteristic of an AML blast phenotype. Given the aggressive nature of this model, we sought to determine whether Npm1 cA;Idh2 R140Q;Flt3 ITD AML cells could be adoptively transferred into either immunodeficient or fully immunocompetent recipients without prior preconditioning. NOD- Prkdc em26Cd52Il2rg em26Cd22/NjuCrl (NCG) and B6.SJL-Ptprca Pepcb/BoyJ (PepBoy) recipients were injected with 1x10 6 bulk splenocytes via the tail vein. Recipient mice quickly develop a lethal AML that faithfully recapitulates the disease observed in the primary transgenic donors. Circulating leukemia cells are detectable from approximately 2 weeks post adoptive transfer (post-AT) and median survival of PepBoy recipients ranged from 17 to 60 days depending on donor (Figure 1). Comprehensive immune profiling using a 36-specificity spectral flow panel showed that in PepBoy recipients, the donor derived cells accurately recapitulate the diversity of the AML immunophenotype of the primary transgenic donor. Notably, cells from male donors do not establish detectable disease in immunocompetent female recipients. This is associated with an increased number of T cells in the female recipients of male cells suggesting immune mediated control of AML development. To assess the response to targeted therapies in a fully immune competent model, we adoptively transferred of our Npm1 cA;Idh2 R140Q;Flt3 ITD to into PepBoy recipients. Mice were randomized to treatment groups based on peripheral blood disease burden at 14 days post AT (0.2-1% of CD45+ cells). Mice received daily oral gavage of either vehicle, 100 mg/kg venetoclax (BCL2 inhibitor), 100 mg/kg enasidenib (IDH2 inhibitor) or 30 mg/kg gilteritinib (FLT3 inhibitor). All three drugs resulted in a significant reduction in circulating tumor burden after 14 days of treatment. Enasidenib did not prolong survival compared to vehicle whereas venetoclax treatment resulted in a significant survival advantage (median 47 days vs 40 in the vehicle). This model was most sensitive to gilteritinib with a median survival of 68 days. (Figure 2). Together these findings demonstrated that the Npm1cA;Idh2 R140Q;Flt3 ITD model is an aggressive model of AML. Adoptive transfer of splenocytes into either immunodeficient NCG or fully immunocompetent PepBoy recipients faithfully recapitulates the disease of primary mice in a reproducible manner. This model therefore provides an in vivo model conducive to drug development studies and provides the opportunity to understand the impact of both AML development and therapeutics on the normal immune system.

UTERINE GRANULOCYTIC SARCOMA AS AN EXTRAMEDULLARY RELAPSE OF ACUTE MYELOID LEUKAEMIA IN AN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENT

Myeloid Sarcoma (MS) is a common condition characterized by proliferation of immature myeloid cells in extramedullary sites. It most often develops in patients with Acute Myelogenous Leukaemia (AML), myeloproliferative neoplasms or myelodysplastic syndromes. MS in AML may precede the disease, appear during the onset or as a relapse, especially in patients after bone marrow transplantation (BMT). Although MS can present in various parts of the body, the most common sites are lymph nodes, central nervous system, bones and soft tissues. MS of the gynaecologic tract is rare, especially in the cervix and uterus. We report an unusual case of EM relapse in the uterus without evidence of a bone marrow (BM) recurrence, two years after an allogeneic BMT. A nulliparous female 32 years old patient was currently attending the hematologic service at a quaternary Brazilian Naval Hospital since 2018, due to AML. She had no specific molecular mutation in the diagnosis. The patient was submitted to an unrelated BMT after her second complete remission in 2020 and had a relapse nine months after, with loss of variable number of tandem repeats (VNTR) from the donor and minimum residual disease (MRD) positive. Donor's Lymphocytes were infused twice and chemotherapy with a hypomethylating agent and BCL-2 inhibitor Venetoclax was established. The patient achieved her third complete remission and was in follow-up until she presented herself with complains of neuropathic and acute abdominal pain. Clinical examination revealed several soft tissue tumorations resembling MS. During the investigation, an abdominal pelvic magnetic resonance imaging (MRI) peculiarly demonstrated a large uterine mass with compression of the right ureter and pyelocalyceal dilation. Gynaecological clinical exam showed a large violaceus mass about four cm with anterior and right vaginal wall infiltration. The first diagnostic hypothesis was primary cervical cancer stage IV. The biopsy revealed a massive infiltration of immature myeloid cells with the expression of anti-ERG and myeloperoxidase antibodies, confirming MS. The immunophenotypical analysis of the bone marrow aspirates showed the patient still had a complete remission with MRD negative and a VNTR with full donor chimerism. The patient started systemic chemoterapy with a hypomethylating agent and BCL-2 inhibitor Venetoclax. Decision making on the treatment of cervical MS is challenging due to the absence of gynaecological classification guidelines. In patients in this age group with no offspring, the choice of therapy should consider the fertility issue, covering from hysterectomy to radiation, with or without a second BMT. Finally, MS should be a differential diagnosis in a patient with a uterine mass and a previous medical history of AML and BMT.

Interleukin 1α (IL-1α) Promotes Pathogenic Immature Myeloid Cells and IL-1β Favors Protective Mature Myeloid Cells During Acute Lung Infection.

Bacterial pneumonia is a common risk factor for acute lung injury and sepsis-mediated death, but the mechanisms underlying the overt inflammation and accompanying pathology are unclear. Infiltration of immature myeloid cells and necrotizing inflammation mediate severe pathology and death during pulmonary infection with Francisella tularensis. However, eliciting mature myeloid cells provides protection. Yet, the host factors responsible for this pathologic immature myeloid cell response are unknown. Here, we report that while the influx of both mature and immature myeloid cells is strictly MyD88 dependent, the interleukin 1 (IL-1) receptor mediates an important dual function via its ligands IL-1α and IL-1β. Although IL-1β favors the appearance of bacteria-clearing mature myeloid cells, IL-1α contributes to lung infiltration by ineffective and pathologic immature myeloid cells. Finally, IL-1α and IL-1β are not the sole factors involved, but myeloid cell responses during acute pneumonia were largely unaffected by lung levels of interleukin 10, interleukin 17, CXCL1, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor.

ITOC2 – 008. Melanoma differentiation-associated protein 5 (MDA5)-based tumour immunotherapy reprograms M2/G2-polarised myeloid-derived suppressor cells (MDSCs)

Background Pancreatic cancer shows a dense infiltration of immature myeloid cells including myeloid-derived suppressor cells (MDSCs). In the tumour, MDSCs polarise in alternatively activated M2/G2 cells, suppressing T cell responses. We previously reported that RNA-based immunotherapy with ligands for the cytosolic helicase MDA5 leads to type I interferon (IFN)-driven tumour immune control in mice with pancreatic cancer. Here we investigated the impact of MDA5 on MDSC functional profiles. Methods Tumour bearing mice were injected intravenous (i.v.) with poly(I:C)-PEI. G-MDSCs (CD11b+Gr-1high) and M-MDSCs (CD11b+Gr-1intLy6C+) from tumours and spleen were isolated for flow cytometry, gene expression analysis and suppression assays. Therapeutic efficacy was assessed in orthotopic tumour models (Panc02, T110299). Results Tumour MDSCs exhibit an immunosuppressive phenotype with high levels of PD-L1, TGF-b, vascular endothelial growth factor (VEGF) and arginase. MDA5 ligands significantly reduced expression levels of TGFb, arginase, Adam17, galectin-9, as well as STAT3 and STAT5b, indicative of a switch from M2/G2 towards M1/G1 differentiation. This correlated with reduced suppressive function in T cell assays. Moreover, treatment led to MDSC apoptosis in vivo and significantly prolonged survival in mice with pancreatic cancer. Conclusion Functional reprograming of MDSCs with MDA5 ligands is a promising strategy for counteracting immunosuppression in pancreatic cancer.

Isolated Myeloid Sarcoma of the Maxillary Sinus: A Case Report and Review of Literature

Myeloid sarcoma is a rare malignant disease defined as extramedullary infiltration of immature myeloid cells and may precede or occur synchronously with acute myeloid leukemia. We reported a 35-year-old male of isolated myeloid sarcoma who presented with symptoms similar to chronic rhinosinusitis initially. Isolated myeloid sarcoma in the paranasal sinuses is exceedingly rare after reviewing the previous literatures. Through proper histological, adequate panels of immunohistochemical stain (positive markers including CD68/KP1, myeloperoxidase, CD 117, CD 99, CD68/PG-M1, lysozyme, CD34, terminal deoxynucleotidyl transferase and negative markers including CD3, CD20, CD45RO and CD79a) and cytogenic study lead to accurate diagnosis. Early intervention with systemic chemotherapy with cytarabine-based regimens is the treatment of choice. The role of chromosomal aberrations and genetic abnormality related to prognosis remain uncertain.

Tumor-Initiated Inflammation Overrides Protective Adaptive Immunity in an Induced Melanoma Model in Mice

We studied the effect of the immune system on two differentially aggressive melanomas developing in mice on conditional deletion of the INK4A/ARF tumor suppressor gene, with concomitant expression of oncogene H-Ras(G12V) and a natural cancer-germline tumor antigen (TA). "Slow progressor" melanomas contained no activated T lymphocytes (TL). In contrast, "aggressive" melanomas were infiltrated by activated TLs lacking effector molecules and expressing high levels of PD-1, indicating an exhausted phenotype. Aggressive melanomas were also infiltrated by immature myeloid cells (IMC). Infiltration was associated with local inflammation and systemic Th2/Th17-oriented chronic inflammation that seemed to impair further activation of TLs, as tumor-specific T cells adoptively transferred into mice bearing aggressive melanomas were poorly activated and failed to infiltrate the melanoma. This immunosuppression also led to the incapacity of these mice to reject inoculated TA-positive tumors, in contrast to slow-progressing melanoma-bearing mice, which were responsive. To test the role of adaptive immunity in tumor progression, we induced melanomas in immunodeficient RagKO compound mice. These mice developed aggressive but not slow-progressing melanomas at a higher frequency and with a shorter latency than immunocompetent mice. Immunodeficient mice also developed abnormal inflammation and infiltration of IMCs in a manner similar to immunocompetent mice, indicating that this phenotype was not dependent on adaptive immunity. Therefore, tumor-intrinsic factors distinguishing the two melanoma types control the initiation of inflammation, which was independent of adaptive immunity. The latter delayed development of aggressive melanomas but was overridden by inflammation.

Chemokine receptors in cancer metastasis and cancer cell‐derived chemokines in host immune response

The chemotactic cytokines called chemokines are a superfamily of small secreted cytokines that were initially characterized through their ability to prompt the migration of leukocytes. Attention has been focused on the chemokine receptors expressed on cancer cells because cancer cell migration and metastasis show similarities to leukocyte trafficking. CXC chemokine receptor 4 (CXCR4) was first investigated as a chemokine receptor that is associated with lung metastasis of breast cancers. Recently, CXCR4 was reported to be a key molecule in the formation of peritoneal carcinomatosis in gastric cancer. In the present review, we highlight current knowledge about the role of CXCR4 in cancer metastases. In contrast to chemokine receptors expressed on cancer cells, little is known about the roles of cancer cell-derived chemokines. Cancer tissue consists of both cancer cells and various stromal cells, and leukocytes that infiltrate into cancer are of particular importance in cancer progression. Although colorectal cancer invasion is regulated by the chemokine CCL9-induced infiltration of immature myeloid cells into cancer, high-level expression of cancer cell-derived chemokine CXCL16 increases infiltrating CD8(+) and CD4(+) T cells into cancer tissues, and correlates with a good prognosis. We discuss the conflicting biological effects of cancer cell-derived chemokines on cancer progression, using CCL9 and CXCL16 as examples.

Reduction in PU.1 activity results in a block to B-cell development, abnormal myeloid proliferation, and neonatal lethality

It has been demonstrated that high concentration of the transcription factor PU.1 (encoded by Sfpi1) promotes macrophage development, whereas low concentration induces B-cell development in vitro. This has led to the hypothesis that lower levels of PU.1 activity are required for B cell than for macrophage development in vivo. We utilized an allele of Sfpi1 (termed BN) with a mutation in the first coding exon, which resulted in a reduction of PU.1 expression in order to test this hypothesis. Using gene targeting in embryonic stem cells, two ATG-start site codons of PU.1 were mutated, resulting in reduced PU.1 expression originating from a third start codon. Mice were assayed for phenotypic abnormalities using fluorescence-activated cell sorting, microscopy, and colony-forming ability. In addition, isolated cells were tested for their differentiation potential in vitro and in vivo. Lymphoid and myeloid cells derived from cultured Sfpi1(BN/BN) fetal liver cells had reduced levels of PU.1 expression and activity. B-cell development was intrinsically blocked in cells isolated from Sfpi1(BN/BN) mice. In addition, myeloid development was impaired in Sfpi1(BN/BN) fetal liver. However, neonatal Sfpi1(BN/BN) mice had a dramatic expansion and infiltration of immature myeloid cells. Contrary to our original hypothesis, high levels of PU.1 activity are required to induce both myeloid and B-cell development. In addition, neonatal mice homozygous for the hypomorphic allele acquire a myeloproliferative disorder and die within 1 month of age.

Cholecystitis Caused by Infiltration of Immature Myeloid Cells: A Case Report

A 59-year-old man with myelodysplastic syndrome who was hospitalized for evaluation of fever and generalized fatigue had elevated levels of C-reactive protein and pancytopenia. A search for a site of infection and empiric treatment with antibiotics were unsuccessful. Over 5 to 6 weeks right upper quadrant pain and rebound tenderness developed. Sonographic Murphys sign was present. Computed tomography showed thickening of the gallbladder wall, and repeated ultrasonography demonstrated changes consistent with cholecystitis. Open cholecystectomy was performed as an emergency procedure. Macroscopically the resected gallbladder showed an edematous and thickened wall. Histopathologic examination revealed transmural infiltration by atypical mononuclear cells with distinct nuclei. The cells showed immunohistochemical staining for CD15, indicating myeloid lineage. By 10 days after surgery, counts of leukocytes and leukoblasts had markedly increased, reaching 36,700/microL and 76.0%, respectively. The blast crisis was thought to indicate progression from myelodysplastic syndrome to leukemia. The patient died of progressive disease 12 days after surgery. We have described a rare case of acute cholecystitis caused by infiltration of immature myeloid cells to the gallbladder. An acute abdomen complicating hematologic disorders is life-threatening and requires prompt and appropriate treatment.