Novel Circulating and Tissue Monocytes as Well as Macrophages in Pancreatitis and Recovery

Abstract

Background and aims: Acute pancreatitis (AP) is an inflammatory disease with mild to severe course that is associated with local and systemic complications and significant mortality. Uncovering inflammatory pathways that lead to progression and recovery will inform ways to monitor and/or develop effective therapies.

Methods: We performed single-cell mass Cytometry by Time Of Flight (CyTOF) analysis to identify pancreatic and systemic inflammatory signals during mild AP (referred to as AP), severe AP (SAP), and recovery using 2 independent experimental models and blood from patients with AP and recurrent AP. Flow cytometric validation of monocytes subsets identified using CyTOF analysis was performed independently.

Results: Ly6C⁺ inflammatory monocytes were the most altered cells in the pancreas during experimental AP, recovery, and SAP. Deep profiling uncovered heterogeneity among pancreatic and blood monocytes and identified 7 novel subsets during AP and recovery, and 6 monocyte subsets during SAP. Notably, a dynamic shift in pancreatic CD206⁺ macrophage population was observed during AP and recovery. Deeper profiling of the CD206⁺ macrophage identified 7 novel subsets during AP, recovery, and SAP. Differential expression analysis of these novel monocyte and CD206⁺ macrophage subsets revealed significantly altered surface (CD44, CD54, CD115, CD140a, CD196, podoplanin) and functional markers (interferon-γ, interleukin 4, interleukin 22, latency associated peptide-transforming growth factor-β, tumor necrosis factor-α, T-bet, RoRγt) that were associated with recovery and SAP. Moreover, a targeted functional analysis further revealed distinct expression of pro- and anti-inflammatory cytokines by pancreatic CD206⁺ macrophage subsets as the disease either progressed or resolved. Similarly, we identified heterogeneity among circulating classical inflammatory monocytes (CD14⁺CD16^-) and novel subsets in patients with AP and recurrent AP.

Conclusions: We identified several novel monocyte/macrophage subsets with unique phenotype and functional characteristics that are associated with AP, recovery, and SAP. Our findings highlight differential innate immune responses during AP progression and recovery that can be leveraged for future disease monitoring and targeting.

Keywords: Acute Pancreatitis; CyTOF; Macrophages; Monocytes; Severe Acute Pancreatitis.

Figure 1:. CyTOF analysis of mouse pancreas during AP and recovery.

(A) Experimental set up for induction of AP and recovery and CyTOF analysis of pancreas and blood. (B) UMAP plot showing various immune cell clusters in the pancreas. (C, D) Volcano plot showing differential abundance analysis of immune cells at all time points (12h, 24h, 48h, 168h) compared to control (0h) and pair wise comparison between 0h vs 12h (AP) respectively. Monocytes were labeled as the top altered immune cell followed by CD206⁺ macrophages. (E) UMAP plot showing clusters of monocytes (red circle) and CD206⁺ macrophages (blue circle) along with the kinetics of AP and recovery phase. (F, G) Frequency of monocytes and CD206⁺ macrophages in the pancreas. (H, I) UMAP plots showing seven novel monocyte subsets and their frequency. (J) DE analysis of monocyte MHCII^loLy6Gc^loCD45RB^lo revealed PDPN, CD140a and CD54 were significantly altered during the AP and recovery. Mo- Monocytes; CD206⁺MØ - CD206⁺ macrophages

Figure 2.. CyTOF analysis of mouse pancreas during CDE diet-induced SAP.

(A) Experimental set up for induction of SAP and CyTOF analysis of pancreas and blood. (B) UMAP plot showing various immune cell clusters in the pancreas. (C) Volcano plot showing differential abundance analysis of immune cells at all the time point comparisons (24h, 48h, 72h) with control mice (0h). Monocytes appeared as the top altered immune cell. (D) UMAP plot showing monocytes cluster (red circle) and CD206⁺ macrophages cluster (blue circle) along with the kinetics of SAP development. (E, F) Dot plot showing frequency of monocytes and CD206⁺ macrophages in the pancreas. (G, H) UMAP plots showing six novel monocyte subsets and their frequency. (I) DE analysis of monocyte MHCII^loLy6Gc^hiCD45RB^lo revealed reduced expression of CD54, CD140a, CD196, PDPN, TNF-α, LAP-TGF-β, T-bet, RoRγt; whereas, (J) monocyte MHCII^hi Ly6Gc^hi showed reduced expression of CD196, and TNF-α during SAP. (K) DE analysis of monocyte subset MHCII^lo Ly6Gc^loCD45RB^lo revealed induced expression of CD54 at 48h and then reduced at 72h during SAP. Mo- monocytes; CD206⁺ MØ- CD206⁺ macrophages

Figure 3:. Novel subsets of pancreas CD206+ macrophages during AP and recovery.

(A, B) UMAP plot showing seven novel subsets of CD206⁺ macrophage and their frequency during AP and recovery. (C) DE analysis of CD206⁺ macrophage subset MHCII^hiLy6Gc^loCD44^hi revealed increased expression of CD54, PDPN, and IL-22 at 24h; whereas (D) subset MHCII^hiLy6Gc^loCD44^lo with increased expression of CD54 during AP (12h) which return to baseline during recovery (168h). (E) DE analysis of CD206⁺ macrophage subset MHCII^lo Ly6Gc^lo CD44^lo display induced expression of CD54 and PDPN (12–24h); whereas (F) subset MHCII^loLy6Gc^loCD44^hi revealed increased expression of PDPN and LAP-TGF-β at 24h which returned to base line during recovery phase (168h).

Figure 4:. Novel circulating monocyte subsets during AP and recovery.

(A) UMAP analysis demonstrating the changes in the cluster of circulatory monocytes cluster (red circle) during AP and recovery. (B) Peak induction in circulatory monocyte was observed at 12h which recover to base line at 168h. (C, D) Seven distinct subsets of monocytes identified by unbiassed profiling and their frequencies. (E) DE analysis of monocyte subset MHCII^lo Ly6Gc^loCD45RB^lo with altered expression of CD115, PDPN, LAP-TGF-β, CD44, and CD54; whereas (F) monocyte subset MHCII^lo Ly6Gc^hiCD45RB^lo demonstrated altered expression of IFN-γ and CD54 during AP and recovery. (G) DE analysis of monocyte subset MHCII^lo Ly6Gc^loCD45RB^hi showed significantly altered expression of CD54, PDPN, and T-bet; whereas (H) monocyte subset MHCII^hi Ly6Gc^loCD45RB^lo revealed significant alterations in expression of CD44, PDPN, CD140a, and IL-4 during AP and recovery.

Figure 5:. Flow cytometric validation of novel monocyte subsets during AP and recovery.

(A) Experimental setup for AP and recovery phase and flowcytometric analysis of pancreas and blood (B) FACS plot showing gating scheme for identification eight different monocyte subsets in the pancreas (numbered as monocyte 1–8). (C) Stacked plot showing frequency of monocyte subsets in the pancreas and (D) Eight subsets of monocytes were identified in circulation during AP and recovery. (E) Stacked plot showing frequency of circulatory monocyte subsets. (F) P values for monocyte subsets during AP and recovery ns=non significant.

Figure 6:. CyTOF analysis of circulating monocytes in pancreatitis patients.

(A) Experimental setup for CyTOF analysis in the blood collected from AP (n=12) and RAP (n=11) patients (B) UMAP analysis of human PBMC indicates a significant increase in classical inflammatory monocytes (CD14⁺ CD16⁻) compared to nonclassical (CD14⁻ CD16⁺) and intermediate (CD14⁺CD16⁺) monocytes in both AP and RAP patients. (C) Frequency (%) of each monocyte subset from AP and RAP is shown. (D, E) Unbiased profiling identified six subclusters of classical monocytes CD14⁺CD16⁻ in circulation of AP and RAP patients and their % frequency.

Figure 7:. Summary of novel monocyte and macrophage subsets in experimental and human pancreatitis.

*Monocyte and CD206⁺ macrophage subsets with differentially expressed phenotypic and functional markers after FDR correction. ^#Monocyte subset identified in blood and pancreas in mild AP and recovery but not in SAP. AP-Acute Pancreatitis; SAP- Severe AP; RAP- Recurrent AP.