Investigating the Impact of IL-17 and IL-4 on Haematopoietic Stem Cells
Date
2023-11-24
Journal Title
Journal ISSN
Volume Title
Publisher
Saudi Digital Library
Abstract
Introduction: Haematopoiesis allows blood cells renewal for maintaining blood’s steady-state and stressadapted
functioning during infections and inflammations. It is driven by haematopoietic system cells
(HSCs) primarily originated at bone marrow. HSCs respond to virus and bacteria-triggered inflammations
through altered proliferation and/or differentiation. Little has been known regarding HSCs functioning at
type-2 and type-17 immune responses associated with helminths and extracellular fungal infections,
respectively. Study aims: We aimed to assess whether specific HSC population (ESLAM) are responsive
to IL-17 ± IL-4 and how this could be translated through ESLAM’s proliferation and differentiation.
Methods: ESLAM HSCs (lineage- c-kit+ Sca1+ CD48– CD150+ EPCR+) were purified from bone marrow
of naïve mice and their proliferation and differentiation -/+ IL-4 and IL-17 were assessed in single cell
liquid cultures and bulk colony formation unit (CFU) assays. Results: IL-4 reduced ESLAMs self-renewal
through single cell cultures, whereas IL-4 increased ESLAM’s differentiation into megakaryocytes. Only
IL-4 altered % differentiated ESLAMs for Ly6C/Ly6G/MHCII markers affecting %
monocytes/macrophage lineage. In CFU, IL-4 reduced ESLAM’s colonies which was then partially
antagonised on IL-17 co-treatment. ESLAMs’ differentiation was increased with cytokine treatments for
Ly6C/Ly6G markers, rather than MHCII, at CD41– CD11b+ subpopulation. Conclusion: cytokines
influence ESLAM’s active self-renewal and differentiation with profound activity for IL-4/Th2 over IL-
17/Th17 immune responses. Future work will involve further ESLAM differentiation analysis under
interferon-γ, type-1 interferon, and tumour necrosis factor-α, and how their responses would be altered by
IL-17 co-treatments. Wider range of ESALM’s
Description
Application of HSC single cell cultures and CFU assay at murine bone marrow has been identified
useful for investigating HSCs proliferation and differentiation under inflammatory-induced conditions
(Chen et al, 2017; Khoramian Tusi & Socolovsky, 2018; Rodríguez et al, 2021; Smajilagić et al, 2013).
Our study highlighted the potential role of IL-4 for reducing ESLAMs self-renewal across single cell culture
and in presence of growth factors relying on c-Kit+/– Sca1+/– markers. Moreover, this Th2 immune mediator
increased ESLAM’s differentiation towards megakaryocyte and myeloid lineages based on CD41, CD11b,
Ly6G/C, and/or MHCII markers. Both IL-4 actions were not influenced by its combination with IL-17
escalated concentration treatments. Findings from CFU study demonstrated the reducing potentiality for
IL-4 on ESALM’s colony counts within MethoCult® culture the thing that was relatively antagonised on
the combination with Th17 immune mediator. Both IL-4 and IL-17 showed no impact on ESLAMs’ myeloid
differentiation regarding MHCII marker within CD41– CD11b+ subpopulation as well as for CD45 CD11b
(microglia). However, ESLAM’s differentiation was decreased for plasmacytoid dendritic cells (CD41–
CD11b– MHCII+) on sole IL-17 treatment, whereas differentiation increased for monocytes/macrophages,
eosinophils, and neutrophils based on Ly6G/C marker analysis at CD41– CD11b+ cells.
Within our adopted HSC single cell culture assay, selecting media components for colony ESLAM
cultures was rationalized for affording adequate large expansion of HCSs as per reported studies. Applying
IL-11 and stem cell factor cytokines were reported optimum for preferentially induce proliferation of longterm
HSC over short-term ones (Feng et al, 2005; Walenda et al, 2011; Yamamoto et al, 2013; Yamamoto
et al, 2018). FBS was present within the HSC media for providing factors for cell attachment, hormones,
and nutrients for the culture cell growth, as well as serving as a buffering agent against media disruptions
such as pH alterations and endotoxin productions (Fang et al, 2017). Both Streptomycin and penicillin
supplements were added for preventing bacterial contaminations for the cell cultures. On the other hand,
insulin/transferrin was applied for reducing toxic levels of peroxides and oxygen radicals as well as
minimizing the FBS amount being needed for low-density adherence and routine maintenance of culture
cells (Ghasemi et al, 2019). Regarding cell culture energy supply, the L-glutamine was used for promoting
high-growth yields and efficient energy metabolism, with no detrimental impacts of excessive ammonia
production on culture cells (Rubin, 2019). Finally, 2-beta mercaptoethanol aids the culture cells to utilize
the cultural nutrient cysteine amino acid owing to the earlier potent reducing potentiality (Bannai, 1992).
On the other hand, MethoCult® was optimal for CFU assays. The methyl-cellulose gel-nature media
comprised of Methylcellulose in Iscove’s medium, FBS, bovine serum albumin (BSA), Iron saturated
human transferrin, 2-mercaptoethanol, stem cell factor, IL-3, IL-6, human erythropoietin (EPO), and
supplements, which were reported beneficial for promoting the optimal differentiation and growth of HSCs
within culture (Yang et al, 2011).
Impact on IL-4 on HSCs has been highlighted in several studies supporting its activity within the
bone marrow niche microenvironment. Within murine systems, IL-4 was reported to be incapable of
supporting significant HSC colony formations (Rennick et al, 1987; Snoeck et al, 1993; Sonoda et al, 1990
Vellenga et al, 1990). This was consistent in our study where sole IL-4 treatments lacked effects on % of c-
Kit Sca1 populations. The Sca1 surface protein has been reported as useful markers for labelling immature
HSCs being presented within long-term/short-term HSCs as well as MPPs (Cheng et al, 2020).
Nevertheless, our findings of reduced HSC self-renewal based on declined c-Kit+ Sca1– with IL-4 + IL-17
highlights the synergistic effect of combined cytokines.
IL-4 impacts on HSC differentiation was highlighted within literature where IL-4 stimulated
neutrophil colony formations while as supressed macrophage colonies at co-treatments with IL-3 and/or
myeloid and granulocyte-colony stimulation factors (Ferrajoli et al, 1993; Sonoda, 1994). In terms of
boosted neutrophil colonies, this was consistent with our study as CD11b+ Ly6C+ Ly6G+ population were
increased at IL-4/IL-17 co-treatments. Regarding macrophage/monocyte lineage, our findings were not
consistent with reported CFU analysis where increased cell counts for CD11b+ Ly6C–/+ Ly6G– at IL-4/IL-
17 co-treatments were depicted. Thus, our results suggest differential mediating effects for IL-17 as
compared to other cytokines for influencing IL-4’s co-impacts on differentiated myeloid lineage. The
Ly6G/C proteins has been reported useful for identifying different myeloid lineage
(eosinophils/neutrophils) and monocyte/macrophage subsets (mature versus immature) (Rose et al, 2012;
Tam et al, 2014). Macrophages/monocytes are identified for CD11b– Ly6C–/+ Ly6G–, whereas CD11b+
Ly6C– Ly6G– and CD11b+ Ly6C+ Ly6G+ for eosinophils and neutrophils, respectively (Misharin et al,
2013).
We recognized prominent decreased immature monocyte/macrophage differentiation over mature
ones (CD11b+ Ly6C–/+ Ly6G– MHCII+) upon IL-4/IL-17 co-treatments. Nevertheless, these findings were
for single cell culture which were different from MethoCult® culture and literature reported CFU analysis.
It worth noting that we included MHCII+ within single cell culture analysis this may impact the CD11b
Ly6C/G expression findings since MHCII-positive cells showed great expression heterogenicity. MHCII
surface marker is generally expressed at various myeloid and lymphoid lineage cells including dendritic
cells, monocytes/macrophages, and lymphocytes the thing that inherit great expression heterogenicity with
HSC’s CD11b–/+ MHCII+ populations (Patente et al, 2018). MHCII-associated heterogenicity was obvious
within our findings the thing that redeemed FACS analysis for the marker’s median expressions (MFI)
(Weisberg et al, 2015). Such heterogenicity could partially explain why we could not find significant impact
for IL-4 ± IL-17 treatments at CD11b–/+ CD41–/+ MHCII+ populations. Concerning microglia
subpopulations (CD45+ CD11bvar and CD45– CD11b–), our results showed no impact of IL-4 ± IL-17 on
the ESLAMs’ differentiation into these macrophage-type subclasses. This would suggest the lack of IL-4 ±
IL-17 impact on generating the yolk sac-type macrophages, the microglia progenitor cells being developed
within the first wave of primitive haematopoiesis (Ginhoux et al, 2010).
The role of IL-17 within haematopoiesis, and particularly in granulopoiesis, has been highlighted
through early reports as this CD4+/Th7 immune signalling protein supports the MPPs growth (Najafi et al,
2021). Schwarzenberger et al. demonstrated profound activation of splenic and bone marrow granulopoiesis
at low-moderate IL-17 concentrations (10 to 100 ng/ml) that in turn caused myeloid HSCs and granulocyte
macrophage MPPs expansions as well as neutrophilia in transgenic murine models (Schwarzenberger et al,
1998). These results were recapitulated through several in vitro and in vivo studies (Jovcić et al, 2007;
Jovcić et al, 2004; Jovcić et al, 2001; Schwarzenberger et al, 2000). To our delight, literature findings are
in agreement with our CFU assay where IL-17 ± IL-4 showed ESLAM’s differentiation skewness towards
the myeloid lineage. IL-17 action has been reported to be highly reliant on the microenvironment niche and
animal status where under stress, the cytokine showed altered impacts on granulopoiesis being different
from normal conditions (Bugarski et al, 2006; Ilić et al, 2010; Schwarzenberger & Kolls, 2002).therefore,
evidence consider IL-17 as distress responder rather than baseline homeostatic factor. In our study we
adopted low to moderate IL-17 concentrations (1 to 100 ng/ml) since several reports showed that IL-17
impact on HSC and MMP could be altered relying on the dose. High IL-17 doses have illustrated inhibitory
actions on granulocyte-macrophage progenitors (Mojsilović et al, 2015). Literature evidence rationalized
our findings for positive impact of IL-17 ± IL-4 on murine bone marrow ESLAM’s differentiation with
preferentiality towards the myeloid lineage .
Herein, we provide evidence for positive impact of IL-17 ± IL-4 on ESLAM differentiation through
single cell culture and CFU analysis. Profound activity was assigned for IL-4/Th2 signalling mediator over
IL-17/Th17. Skewness towards ESLAMs’ differentiated myeloid lineage has also been presented. Our
findings further highlighted the superiority of Ly6C/G surface marker over MHCII for identifying the
ESLAM’s differentiated myeloid lineage under the designated cytokine culture treatments. Owing to the
heterogenic activity of IL-17, future work will involve further ESLAM differentiation analysis in
combination with other Th immune-inflammatory mediators. Interferon-γ, type-1 interferon, and tumour
necrosis factor-α would be investigated on ESLAM’s differentiations showing how their responses would
be altered on IL-17 co-treatments. Wider range of ESALM’s marker expression analysis would also be
performed for differentiating various myeloid lineage members.
Keywords
IL-17, IL-4, HSCs, immune responses, proliferation and differentiation, inflammations, Haematopoiesis