D Kioroglou, J L Cabriada, U Martinez Marigorta, I Rodriguez-Lago. Journal of Crohn’s and Colitis, Volume 18, Issue Supplement_1, January 2024, Page i389,doi.org=10.1093/ecco-jcc/jjad212.0236

Background
Granulocyte-monocyte apheresis (GMA) is a non-pharmacological treatment approved for the management of ulcerative colitis (UC), particularly steroid-dependent cases. The exact mechanism of action and immunological changes associated with GMA remain undescribed. Gene expression analysis at the single-cell level (through scRNA-Seq) has emerged as a key tool of choice to characterize drug response at the molecular level. We used scRNA-Seq to characterize the transcriptomic effects and immune cell population alterations in GMA.

Methods
We generated scRNA-Seq from peripheral blood mononuclear cells (PBMC) of two ulcerative colitis (UC) patients undergoing GMA treatment. We compared the gene expression profile before and after 5 sessions of GMA treatment. The analytical pipeline included quality control and classical filtering steps, cell-type annotation, differential gene expression analysis and pathway enrichment profiling.

Results
We report three main results. First, we observed significant reductions of cell types directly affected by GMA treatment in UC patients. This includes classical CD14+ monocytes and Natural killers, which are central components of the innate system. Of note, we observed a remarkable increase in Double-negative T cells (dnT) after 5 sessions of GMA treatment, suggesting potential expansion of protective populations involved in decreased inflammation. Second, we observe a variety of genes and regulatory pathways altered by GMA treatment. In total, we detect 86 differentially expressed genes (DEGs), which overall are biased towards downregulation (63%). Of note, we detected dnT-s exhibiting upregulation of NEFL that is associated with the MAPK cascade and downregulation of genes related to immune response and signaling pathways. Finally, the effects of GMA treatment extend beyond the above mentioned populations, with particular alterations in CD4+ T cell populations such as CD4+ central memory and CD4+ Naive (7 and 13 DEGs, respectively).

Conclusion
For the first time, we generated single-cell transcriptomic profiles to characterize the effects of GMA treatment in peripheral blood of UC patients. Our preliminary analysis detects important alterations in the gene regulation and cell type composition in samples obtained after 5 sessions of GMA. Through expansion of this dataset to include more time points and profiles for more individuals, we will discuss the longitudinal changes and molecular mechanisms involved in response to GMA treatment.

Ang Cui ^1 2 3, Teddy Huang ⁴, Shuqiang Li ^5 4, Aileen Ma ^5 6, Jorge L Pérez ^5 6, Chris Sander ^5 7 8, Derin B Keskin ^5 4 9, Catherine J Wu ^5 9 10, Ernest Fraenkel ^5 11, Nir Hacohen ^12 13 14

Nature 2024 Jan;625(7994):377-384. doi: 10.1038/s41586-023-06816-9. Epub 2023 Dec 6.

Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets^1-3. A myriad of studies have highlighted their central role in immune function^4-13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine-cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1β induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response.

Esteban Sáez-González MD, Inés Moret-Tatay PhD, Guillermo Bastida MD, PhD, Mariam Aguas MD, PhD, Marisa Iborra MD, PhD, Pilar Nos MD, PhD, Belén Beltrán MD, PhD  J Clin Apher. 2023 38(6); 1-10. doi:10.1002/jca.22101

Background

Ulcerative colitis (UC) is an inflammatory bowel disease characterized by chronic inflammation of the gastrointestinal tract, affecting millions of individuals throughout the world, and producing an impaired health-related quality of life. Granulocyte and monocyte apheresis (GMA) is a therapeutic option for UC management to induce remission by selective removal of activated leukocytes from bloodstream. Despite the knowledge of the important role of epigenetics in UC pathogenesis, and in the response to different treatments, nothing is known about the role of microRNAs in GMA therapy in UC patients.

Methods

7 consecutively UC patients who started GMA in combo therapy with infliximab were recruited. Peripheral blood samples were taken before the apheresis session, at the start of the induction (S0) and at the end (S10). They were follow-up during the induction phase (10 sessions: 2 sessions for a week during 3 wk and 1 session for a week during 4 wk) of the treatment at a tertiary hospital (Hospital la Fe) and 6 mo after finishing the GMA induction therapy. MiRNA was extracted and analyzed by RT-PCR. R software and GraphPad were used.

Results

Clinical disease activity significantly decreased after induction therapy with GMA (median partial Mayo score 2 (IQR, 1-6) (P < .05). Fecal calprotectin value and CRP value significantly decreased after induction therapy. Five microRNAs modified their expression during GMA (unsupervised analysis): miR-342-3p, miR-215-5p, miR-376c-3p, miR-139-5p, and miR-150-5p. When a sub-analysis was performed in those patients who showed good response to apheresis treatment (n = 5), two microRNAs showed to be implicated: miR-215-5p and miR-365a-3p. These are preliminary but promising and novel results, as it is the first time, to our knowledge that microRNA profiles have been studied in the context of GMA treatment for IBD.

MicroRNA and granulocyte-monocyte adsorption apheresis combotherapy after inadequate response to anti-TNF agents in ulcerative colitis – PubMed (nih.gov)

MicroRNA and granulocyte‐monocyte adsorption apheresis combotherapy after inadequate response to anti‐TNF agents in ulcerative colitis – Sáez‐González – Journal of Clinical Apheresis – Wiley Online Library

D. Kioroglu1 , J.L. Cabriada2, U. M. Marigorta1,3, I. Rodríguez-Lago Ueg journal

Volume11, Issue S8 Supplement: 31st United European Gastroenterology Week 2023 October 2023 Pages 799

Introduction: Granulocyte–monocyte apheresis (GMA) is a non-pharmacological therapy approved for the treatment of ulcerative colitis (UC), mainly in steroid-dependent cases. Its mechanism of action is based onthe removal of activated leukocytes, but its exact immunological changes have not been fully described yet. Aims & Methods: Our aim was to characterize the response in the transcriptome at the single-cell resolution level and cell population effects of GMA device. We analysed scRNASeq data from peripheral blood mono-nuclear cells (PBMC) of two UC patients undergoing their first GMA session. Their gene expression profile was compared before (PRE) and one month after (POST) the GMA treatment, from the inflow and outflow lines, respectively. The analytical pipeline included quality control and filtering, cell-type annotation, differential gene expression and pathway enrichment analysis. Results: Two patients with UC (mean age 59 years; both E2) were included.Overall, the cell populations that appear to have been affected by the GMA treatment were natural killer cells and monocytes with both populations being reduced after the treatment. The distribution of the annotated cell-types was 2,369 B cells (PRE:50%, POST:49%), 11,462 CD4+ (PRE:49%,POST:50%) and 8,845 CD8+ T-cells (PRE:51%, POST:48%), 47 dendritic cells (PRE:40%, POST:59%), 3,773 natural killer cells (PRE:54%, POST:45%) and 2,352 monocytes (PRE:59%, POST:40%). Annotation at higher resolution identified 27 cell-types with monocytes being subannotated as 1,972 CD14 (PRE:62%, POST:37%) and 380 CD16 (PRE:48%, POST:51%) monocytes. Differential gene expression analysis identified in total 86 significant genes across six cell-types (CD4+ naive and central memory T-cells, natural killer cells, B intermediate cells, double-negative T-cells and CD14monocytes) with 63% of these genes being downregulated after the GMA treatment. Pathway enrichment analysis identified higher contribution of the double-negative T-cells to the enriching genes. More specifically, after the treatment the double-negative T-cells exhibited upregulation of the NEFL that is associated with the MAPK cascade and downregulation of genes related to immune response and signalling pathways. Regarding the CD14 monocytes, after the GMA treatment we observed significant downregulation of the genes LINC02315, IGHEP1 and IGHE, with the latter being linked to innate immune response pathways. Conclusion: GMA induces a range of modifications in the gene expression profile across different cell types that change the immunological environment of UC patients.

Gustaf Wigerblad ¹, Qilin Cao ¹, Stephen Brooks ², Faiza Naz ³, Manasi Gadkari ¹, Kan Jiang ², Sarthak Gupta ¹, Liam O’Neil ¹, Stefania Dell’Orso ³, Mariana J Kaplan ¹, Luis M Franco ¹ J Immunol 2022 Aug 15;209(4):772-782. doi: 10.4049/jimmunol.2200154.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.