Mesenchymal stem cells as endogenous regulators of leukocyte recruitment; the effects of differentiation

Mesenchymal stem cells (MSC) are a tissue-resident stromal cell population that are able to regulate immune responses, in particular the capacity for endothelial cells (EC) to support leukocyte recruitment. In this thesis we examined the ability of MSC from different sources (bone marrow, Wharton’s jelly and trabecular bone) to regulate neutrophil recruitment to inflamed EC and how these responses are altered upon adipogenic differentiation of MSC. Using two flow based adhesion models with varying degrees of proximity between MSC and EC, we observed that all MSC populations suppressed neutrophil recruitment. IL-6 and TGFβ were identified as common bioactive agents found in all co-cultures. Upon differentiation, MSC exhibited a diminished capacity to suppress neutrophil, but not peripheral blood lymphocyte, recruitment. Loss of suppression by MSC-derived adipocytes was reversed by neutralising IL-6. Adipose tissue-derived mature adipocytes and culture differentiated pre-adipocytes did not recapitulate the effects of MSC-derived adipocytes. These data suggest that crosstalk between tissue-resident MSC and EC, dampens the endothelial response to cytokines and limits the aberrant infiltration of circulating leukocytes during inflammation. Upon adipogenic differentiation, MSC lose this regulatory capacity. This could impact on the beneficial effects of MSC in chronically inflamed sites where aberrant infiltration of leukocyte is a main driver of the disease

Thematic Progression: A Comparative Analysis of Social Science Research Articles Written by Pakistani and British Research Scholars

Cohesion plays a crucial role in shaping the texture of a text through appropriate thematic choices. Recognizing the issue of cohesion, the researcher conducted a present study to address this problem. The study focused on analyzing social science research articles authored by Pakistani and British research scholars, employing thematic terminologies and emphasizing the textual metafunction. The objectives were twofold: first, to explore and compare the thematic meanings expressed in Pakistani and British social science research articles, and second, to elucidate the functional interpretation of the identified themes. The researcher conducted a qualitative analysis of the data in three steps: (1) dividing the text into clauses, (2) identifying theme components, and (3) tagging the components according to their types and sub-types of themes. The results revealed that the prominent sub-types of themes were the unmarked topical theme (UMT), finite, continuatives, and conjunctions in the topical, interpersonal, and textual themes, respectively. The topical themes served as noun groups, while the textual themes connected new information with prior information. The qualitative analysis focused on studying the patterns of cohesion, leading the researcher to thematic progression and explaining the relationship between theme and rheme within a text. This relationship and its interpretation demonstrated that cohesion relies on thematic choices. Furthermore, the study found that British research articles exhibited greater cohesion compared to Pakistani research articles

Cancer-associated fibroblasts induce antigen-specific deletion of CD8 + T Cells to protect tumour cells.

Tumours have developed strategies to interfere with most steps required for anti-tumour immune responses. Although many populations contribute to anti-tumour responses, tumour-infiltrating cytotoxic T cells dominate, hence, many suppressive strategies act to inhibit these. Tumour-associated T cells are frequently restricted to stromal zones rather than tumour islands, raising the possibility that the tumour microenvironment, where crosstalk between malignant and "normal" stromal cells exists, may be critical for T cell suppression. We provide evidence of direct interactions between stroma and T cells driving suppression, showing that cancer-associated fibroblasts (CAFs) sample, process and cross-present antigen, killing CD8+ T cells in an antigen-specific, antigen-dependent manner via PD-L2 and FASL. Inhibitory ligand expression is observed in CAFs from human tumours, and neutralisation of PD-L2 or FASL reactivates T cell cytotoxic capacity in vitro and in vivo. Thus, CAFs support T cell suppression within the tumour microenvironment by a mechanism dependent on immune checkpoint activation

Analyzing the effects of stromal cells on the recruitment of leukocytes from flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro “vascular” models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment. Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy. In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium. Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation

Comparative ability of mesenchymal stromal cells from different tissues to limit neutrophil recruitment to inflamed endothelium

Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton's jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary

Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous immune population with diverse immunosuppressive functions in solid tumors. Here, we explored the role of the tumor microenvironment in regulating MDSC differentiation and immunosuppressive properties via signal-regulatory protein alpha (SIRPα)/CD47 signaling. In a murine melanoma model, we observed progressive increases in monocytic MDSCs and monocyte-derived dendritic cells that exhibited potent T cell-suppressive capabilities. These adaptations could be recapitulated in vitro by exposing hematopoietic stem cells to tumor-derived factors. Engagement of CD47 with SIRPα on myeloid cells reduced their phagocytic capability, enhanced expression of immune checkpoints, increased reactive oxygen species production, and suppressed T cell proliferation. Perturbation of SIRPα signaling restored phagocytosis and antigen presentation by MDSCs, which was accompanied by renewed T cell activity and delayed tumor growth in multiple solid cancers. These data highlight that therapeutically targeting myeloid functions in combination with immune checkpoint inhibitors could enhance anti-tumor immunity

Modelling structural and cellular elements and functional responses to lymphatic-delivered cues in a murine lymph node on a chip

Lymph nodes (LNs) are organs of the immune system, critical for maintenance of homeostasis and initiation of immune responses, yet there are few models that accurately recapitulate LN functions in vitro. To tackle this issue, an engineered murine LN (eLN) has been developed, replicating key cellular components of the mouse LN; incorporating primary murine lymphocytes, fibroblastic reticular cells, and lymphatic endothelial cells. T and B cell compartments are incorporated within the eLN that mimic LN cortex and paracortex architectures. When challenged, the eLN elicits both robust inflammatory responses and antigen-specific immune activation, showing that the system can differentiate between non specific and antigen-specific stimulation and can be monitored in real time. Beyond immune responses, this model also enables interrogation of changes in stromal cells, thus permitting investigations of all LN cellular components in homeostasis and different disease settings, such as cancer. Here, how LN behavior can be influenced by murine melanoma-derived factors is presented. In conclusion, the eLN model presents a promising platform for in vitro study of LN biology that will enhance understanding of stromal and immune responses in the murine LN, and in doing so will enable development of novel therapeutic strategies to improve LN responses in disease

Stromal Amyloid β drives Neutrophil extracellular trap formation to augment tumour growth

Tumors are comprised of cancer cells and a network of non-cancerous stromal cells. Cancer-associated fibroblasts (CAFs) are well known to support tumorigenesis and are emerging as immune modulators. While many leukocyte populations are well studied in cancer, neutrophils have received less attention. Neutrophils can release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NETs) in a process termed NETosis. Here, we show that CAFs induce formation of NETs both within the tumor microenvironment and at systemic levels in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on PAD4 and CD11b. Remarkably, CAF-derived Amyloid β was identified as the key factor driving t-NETosis, a protein with significance in both neurodegenerative and inflammatory disorders. Therapeutic inhibition of NETs in established tumors prevented growth, skewing neutrophils to a pro-inflammatory phenotype. Reciprocally, t-NETs enhanced CAF activation phenotypes. Mirroring murine observations, NETs were detected juxtaposed to CAFs in human melanoma and pancreatic adenocarcinoma, and elevated expression of amyloid and β-Secretase correlated with poor prognosis. In summary, we report the existence of cross-talk between CAFs and neutrophils within the tumour microenvironment whereby CAF-induced t-NETosis supports cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target. Significance This study defines the existence of a pro-tumor immunomodulatory function of the stroma showing the induction of Neutrophil Extracellular Traps through CAF-derived Amyloid β. We term this novel process “Tumor-induced NETosis” (t-NETosis) and propose that therapeutic inhibition of this mechanism, which we observe in human melanoma and pancreatic cancer, has the potential to improve patient outcome

Adipogenic differentiation of MSC alters their immunomodulatory properties in a tissue-specific manner

Abstract Chronic inflammation is associated with formation of ectopic fat deposits that might represent damage-induced aberrant mesenchymal stem cell (MSC) differentiation. Such deposits are associated with increased levels of inflammatory infiltrate and poor prognosis. Here we tested the hypothesis that differentiation from MSC to adipocytes in inflamed tissue might contribute to chronicity through loss of immunomodulatory function. We assessed the effects of adipogenic differentiation of MSC isolated from bone marrow or adipose tissue on their capacity to regulate neutrophil recruitment by endothelial cells and compared the differentiated cells to primary adipocytes from adipose tissue. Bone marrow derived MSC were immunosuppressive, inhibiting neutrophil recruitment to TNFα-treated endothelial cells (EC), but MSC-derived adipocytes were no longer able to suppress neutrophil adhesion. Changes in IL-6 and TGFβ1 signalling appeared critical for the loss of the immunosuppressive phenotype. In contrast, native stromal cells, adipocytes derived from them, and mature adipocytes from adipose tissue were all immunoprotective. Thus disruption of normal tissue stroma homeostasis, as occurs in chronic inflammatory diseases, might drive “abnormal” adipogenesis which adversely influences the behavior of MSC and contributes to pathogenic recruitment of leukocytes. Interestingly, stromal cells programmed in native fat tissue retain an immunoprotective phenotype.</jats:p