Umbilical Cord-Derived CD362+ Mesenchymal Stromal Cells Attenuate Polymicrobial Sepsis Induced by Caecal Ligation and Puncture

Abstract

Mesenchymal stromal cells (MSCs) have a multimodal, immunomodulatory mechanism of action and are now in clinical trials for single organ and systemic sepsis. However, a number of practicalities around source, homogeneity and therapeutic window remain to be determined. Here, we utilised conditioned medium from CD362⁺-sorted umbilical cord-human MSCs (UC-hMSCs) for a series of in vitro anti-inflammatory assays and the cryopreserved MSCs themselves in a severe (Series 1) or moderate (Series 2+3) caecal ligation and puncture (CLP) rodent model. Surviving animals were assessed at 48 h post injury induction. MSCs improved human lung, colonic and kidney epithelial cell survival following cytokine activation. In severe systemic sepsis, MSCs administered at 30 min enhanced survival (Series 1), and reduced organ bacterial load. In moderate systemic sepsis (Series 2), MSCs were ineffective when delivered immediately or 24 h later. Of importance, MSCs delivered 4 h post induction of moderate sepsis (Series 3) were effective, improving serum lactate, enhancing bacterial clearance from tissues, reducing pro-inflammatory cytokine concentrations and increasing antimicrobial peptides in serum. While demonstrating benefit and immunomodulation in systemic sepsis, therapeutic efficacy may be limited to a specific point of disease onset, and repeat dosing, MSC enhancement or other contingencies may be necessary.

Keywords: inflammation; mesenchymal stem cell; sepsis.

Figure 1

Anti-inflammatory capacity of CD362⁺ umbilical cord mesenchymal stromal cells conditioned medium (UC-MSC-CM) in vitro. CD362⁺ UC-MSC-CM decreased interleukin 1β (IL-1β)-induced activation of the nuclear factor kappa B (NF-κB) pathway (A) and enhanced wound closure (B) in pulmonary epithelial cells. CD362⁺ UC-MSC-CM increased the rate of phagocytosis in THP-1 macrophages (C) and reduced the production of IL-6 in peritoneal macrophages in response to lipopolysaccharide (LPS) (D). In other sepsis-relevant tissue cells, CD362⁺ UC-MSC-CM improved viability in kidney-derived HK2 (E) and gut-endothelial-derived T84 cells (F) after cytomix-induced injury. * Statistically significant (p < 0.05) with respect to cytomix group. CD362⁺ UC-MSCs reduce IL-6 peritoneal macrophage production after LPS stimulation (D). * Statistically significant (p < 0.05) with respect to cytomix (A,E,F), vehicle at (B) or vehicle at same LPS concentration (C,D). Columns represent mean (n = 6), error bars represent SD.

Figure 2

Survival after severe caecal ligation and puncture (CLP) injury and CD362⁺ UC-MSC administration. Administration of CD362⁺ UC-MSCs contemporaneous with severe systemic sepsis induction increased mean survival duration (A, n = 32/14) and total survival at the defined 48 h timepoint (B). Bars represent mean (n = 32/14), error bars represent SD.

Figure 3

Reduced serum lactate and bacterial genome number and increased antimicrobial peptide after CD362⁺ UC-MSC administration at 4 h. Administration of CD362⁺ UC-MSCs 4 h after CLP induction reduced the lactate measured in serum 48 h after the CLP procedure compared with vehicle (A). Reduction of copies of the 16S fraction of the bacterial genome in serum after CD362⁺ UC-MSC administration. (B). Reduction of copies of 16S fraction of bacterial genome in peritoneal lavage serum after CD362⁺ UC-MSC administration (C). Reduction of copies of 16S fraction of bacterial genome in the liver after CD362⁺ UC-MSC administration (D). Increased concentration of the antimicrobial peptide hepcidin in serum after CD362⁺ UC-MSC treatment compared with vehicle control (E). Increased presence of the antimicrobial peptide hepcidin in peritoneal lavage in the CD362⁺ UC-MSC treated group compared with vehicle control (F). Bars represent mean (n = 3/8/8), error bars represent SD. * Statistically significant (p < 0.05) with respect to vehicle group.

Figure 4

Bacterial CFU reduction after contemporary CD362⁺ UC-MSC administration in severe systemic sepsis. The colony counts representative of Klebsiella spp. (A), Enterococcus spp. (B) and Escherichia spp. (C) were reduced in liver tissue homogenate at 48 h after CD362⁺ UC-MSC administration in animals who underwent severe systemic sepsis. This was also observed in spleen homogenate samples (D–F). Bars represent mean (n = 8/14), error bars represent SD. * = Statistically significant (p < 0.05) with respect to control.

Figure 5

Systemic and organ inflammatory cytokine production after CD362⁺ UC-MSC administration. Serum levels of CINC-1 (A), KIM-1 (B) and IL-6 (C) are reduced in the CD362⁺ UC-MSC treated group compared with vehicle administration. In the liver, levels of CINC-1 (D), KIM-1 (E) and IL-6 (F) are reduced in the CD362⁺ UC-MSC treated group compared with vehicle administration. Bars represent mean (n = 3/5/5), error bars represent SD. * Statistically significant (p < 0.05).

Figure 6

Other systemic soluble factor expression after CD362⁺ UC-MSC administration. Levels of circulating macrophage colony-stimulator factor (M-CSF) (A), granulocyte-macrophage colony-stimulating factor (GM-CSF) (B), MIP-1α (C) and vascular endothelial growth factor (VEGF) (D) in serum were reduced in the CD362⁺ UC-MSC treated group compared with vehicle administration. Bars represent mean (n = 3/8/8), error bars represent SD. * Statistically significant (p < 0.05) with respect to vehicle control group.