1 s2.0 S0014488622002242 Main

Experimental Neurology 357 (2022) 114199
Contents lists available at ScienceDirect
Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr
Review article
Mesenchymal stromal cell secretome for traumatic brain injury: Focus on

immunomodulatory action
Francesca Pischiutta a, Enrico Caruso a, b, Helena Cavaleiro a, c, d, e, Antonio J. Salgado c, d,
David J. Loane f, Elisa R. Zanier a, *
a
Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Department of Neuroscience, Milan, Italy
b
Neuroscience Intensive Care Unit, Department of Anesthesia and Critical Care, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy
c
Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal
d
ICVS/3B’s - PT Government Associate Laboratory, Braga, Guimarães, Portugal
e
Stemmatters, Biotechnology and Regenerative Medicine, Guimarães, Portugal
f
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland
A R T I C L E I N F O A B S T R A C T
Keywords: The severity and long-term consequences of brain damage in traumatic brain injured (TBI) patients urgently calls
Traumatic brain injury for better neuroprotective/neuroreparative strategies for this devastating disorder. Mesenchymal stromal cells
Mesenchymal stromal cells (MSCs) hold great promise and have been shown to confer neuroprotection in experimental TBI, mainly through
Secretome
paracrine mechanisms via secreted bioactive factors (i.e. secretome), which indicates significant potential for a
Immunomodulation
cell-free neuroprotective approach. The secretome is composed of cytokines, chemokines, growth factors, pro
Ageing
teins, lipids, nucleic acids, metabolites, and extracellular vesicles; it may offer advantages over MSCs in terms of
delivery, safety, and variability of therapeutic response for brain injury.
Immunomodulation by molecular factors secreted by MSCs is considered to be a key mechanism involved in
their multi-potential therapeutic effects. Regulated neuroinflammation is required for healthy remodeling of
central nervous system during development and adulthood. Moreover, immune cells and their secreted factors
can also contribute to tissue repair and neurological recovery following acute brain injury. However, a chronic
and maladaptive neuroinflammatory response can exacerbate TBI and contribute to progressive neuro
degeneration and long-term neurological impairments.
Here, we review the evidence for MSC-derived secretome as a therapy for TBI. Our framework incorporates a
detailed analysis of in vitro and in vivo studies investigating the effects of the secretome on clinically relevant
neurological and histopathological outcomes. We also describe the activation of immune cells after TBI and the
immunomodulatory properties exerted by mediators released in the secretome. We then describe how ageing
modifies central and systemic immune responses to TBI and discuss challenges and opportunities of developing
secretome based neuroprotective therapies for elderly TBI populations. Finally, strategies aimed at modulating
the secretome in order to boost its efficacy for TBI will also be discussed.
Abbreviations: 3-HAA, 3-hydroxyanthranilic acid; 3HK, 3-hydrxykynurenine; ASCs, adipose stem cells; ATP, adenosine triphosphate; BBB, blood brain barrier; BM,
bone marrow; CCI, controlled cortical impact; CHI, closed head injury; COX, cycloxygenase; DAMPs, damage associated molecular patterns; DCs, dendritic cell; EBST,
elevated body swing test; EVs, extracellular vesicles; GDNF, glial cell-derived neurotrophic factor; HMGB1, high-mobility group box1; HSPs, heat shock proteins; ICP,
intracranial pressure; IDO, indoleamine 2,3-dioxygenase; INFγ, interferon-γ; KYN, kynurenine; LPS, lipopolysaccharide; MMPs, matrix metalloproteinases; mNSS,
modified neurological severity score; MPO, myeloperoxidase; MSCs, mesenchymal stromal cells; MWM, Morris water maze; Ngb, neuroglobulin; NLRs, NOD-like
receptors; NOS2, inducible nitric oxide synthase; PD-1, programmed death-1 receptor; PD-L, programmed death ligand; PGs, prostaglandins; PRRs, pattern recog
nition receptors; RAWM, radial arm water maze; ROS, reactive oxygen species; SDF-1, stromal cell-derived factor-1; sPD-L, soluble form of programmed death ligand;
sTNFR1, soluble form of TNFα receptor 1; TBI, traumatic brain injury; TIMP3, tissue inhibitor of matrix metalloproteinase-3; TLRs, toll-like receptors; TNFα, tumor
necrosis factor α; TRP, tryptophan; TSG-6, TNF stimulated gene-6; UC, umbilical cord; WJ, wharton jelly.
* Corresponding author at: Laboratory of Acute Brain Injury and Therapeutic Strategies, Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario
Negri IRCCS, Via Mario Negri 2, 20156 Milan, Italy
E-mail address: elisa.zanier@marionegri.it (E.R. Zanier).
https://doi.org/10.1016/j.expneurol.2022.114199
Received 18 March 2022; Received in revised form 14 June 2022; Accepted 3 August 2022
Available online 8 August 2022
0014-4886/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
F. Pischiutta et al. Experimental Neurology 357 (2022) 114199
1. Introduction 1.1. Experimental data show efficacy of secretome based therapy in TBI
Traumatic Brain Injury (TBI) is a leading cause of death and Our Medline enquire (as per this link, last update in December 2021)
disability worldwide with no neuroprotective treatment available. The screened experimental studies testing the efficacy of MSC-derived
immune system contributes to the long-term sequelae of TBI, with a secretome in TBI. Study eligibility was restricted to research articles in
complex interaction between central and peripheral components leading English, and no restrictions were made based on publication date. 114
to detrimental and/or beneficial consequences (Simon et al., 2017). articles were screened by two authors (E.C. and H. C.) leading to the
Therapeutic strategies targeting neuroinflammation are being devel selection of 5 in vitro and 9 in vivo studies. Main results are described
oped and are primarily focused on limiting the acute pro-inflammatory below and summarized in Tables 1 and 2. We limited our literature
response and promoting anti-inflammatory and pro-regenerative im search on the secretome as a whole, we acknowledge that the use of
mune activation that may resolve chronic neuroinflammation and pro exosomes or extracellular vesicles is becoming increasingly attractive
mote functional recovery. In this context, mesenchymal stromal cells (Zhang et al., 2015, 2021) and we refer the reader to compelling reviews
(MSCs) represent an ideal therapeutic approach. MSCs have been shown on this topic (Cui et al., 2022; Mot et al., 2022; Yang et al., 2017).
to confer significant neuroprotective effects in experimental TBI models Overall, the protocols used to generate the secretome are varied.
and across multiple research laboratories (Pischiutta et al., 2021). MSCs Differences are related to MSC source, with adipose tissue (ASC) being
induce protection after intracerebral or systemic infusion (Pischiutta the most commonly used (Tajiri et al., 2014; Torrente et al., 2014; Kappy
et al., 2016). MSCs provide trophic support against toxic stimuli to the et al., 2018; Jha et al., 2018, 2019; Xu et al., 2020; Jha et al., 2021; Baez-
damaged brain tissue, stimulate endogenous reparative mechanisms (e. Jurado et al., 2018a, 2018b, 2019a) followed by bone marrow (BM)
g. angiogenesis, neurogenesis, synaptogenesis, brain plasticity pro (Chang et al., 2013; Chuang et al., 2012) and umbilical cord (UC) (Liu
cesses), and possess intrinsic immunomodulatory actions (Xiong et al., et al., 2020a; Liu et al., 2020b). The timing for secretome collection
2018). When administered systemically, MSCs are mostly trapped into ranges from 24 (Tajiri et al., 2014; Jha et al., 2018, 2019; Xu et al., 2020;
filter organs (lungs, spleen and liver) where they interact with host cells Jha et al., 2021; Chuang et al., 2012; Chang et al., 2013; Liu et al.,
decreasing inflammation and modulating the activation of immune cells 2020a), 48 (Baez-Jurado et al., 2019a; Baez-Jurado et al., 2018a; Baez-
(Bustos et al., 2013; de Witte et al., 2018; Ko et al., 2016). Protective Jurado et al., 2018b; Torrente et al., 2014) and up to 72 hours (Liu et al.,
effects are obtained even when apoptotic, metabolically inactivated or 2020b). In addition, a variety of preconditioning stimuli were used to
fragmented MSCs are administered (Weiss and Dahlke, 2019), suggest prime MSC reactivity and potentiate the release of bioactive factors.
ing the beneficial effects are at least partially independent from the These include, exposure of MSC to pro-inflammatory cytokines (e.g.
cellular activity of MSCs. All these premises indicates that the interac tumor necrosis factor-α, TNFα; interferon-γ, INFγ) (Jha et al., 2021; Jha
tion between the infused MSCs and the brain injured tissue is not needed et al., 2019; Jha et al., 2018), hypoxia (Chang et al., 2013; Xu et al.,
for the observed protection and the MSC-released factors (i.e. secre 2020) or brain extract from traumatized rodents (Liu et al., 2020a). To
tome) now stand as the major driver of protection and repair, supporting obtain a solution enriched with bioactive molecules, the secretome was
a cell-free approach using the secretome as a therapeutic option for TBI concentrated by ultra-centrifugal filtration devices with molecular
(Baez-Jurado et al., 2019b; Muhammad, 2019; Pinho et al., 2020; weight cut-off ranging from 3-5 kDa (Chang et al., 2013; Chuang et al.,
Teixeira and Salgado, 2020). A meta-analysis on MSC-based therapeutic 2012; Jha et al., 2021; Jha et al., 2019; Jha et al., 2018; Xu et al., 2020)
approaches in TBI preclinical models shows higher improvement in to 100 kDa (Liu et al., 2020a). These cut-offs provide enriched bioactive
neurological function after treatment with cell-free derivatives over molecules based on their molecular weight, but there was no informa
MSC counterpart (Pischiutta et al., 2021). MSCs have shown high tion about the quantity of factors in MSCs and how much were admin
functional heterogeneity related to intrinsic donor features (tissue istered. In fact, the MSC starting material from which secretome was
source, genotype, age, body mass index, lifestyle choices, concomitant collected was rarely reported, as well as the total μg of proteins released
pathophysiologic conditions) or extrinsic culture conditions (method within the administered secretome. There was no consensus on the
chosen to isolate the cells, the composition of the culture media, the type secretome preparation protocols, and therefore, comparing results from
of plastic surface in which they are grown, the number of passages, the different laboratories that employ MSC-derived secretome for TBI
use of static/dynamic processes) leading to significant MSC’s variability therapy is difficult.
and bottlenecks in the clinical translation (Olmedo-Moreno et al., 2022; Results from in vitro TBI studies exploring the secretome effects and
Srinivasan et al., 2022). MSC-derived secretome, can be fully charac potential mechanisms of action are summarized in table 1. The majority
terized through omics approaches. The identification of the molecules of the studies (Baez-Jurado et al., 2019a; Baez-Jurado et al., 2018a,
endowed with protective/regenerative actions would enable the con 2018b; Torrente et al., 2014) are from the same research group and used
struction of a safe, cell-free and standardized therapeutic strategy with human astrocytes-cell line (T98G) exposed to scratch injury (mimicking
direct clinical implications. Moreover, compared to cell therapy, the use the mechanical injury) combined with glucose deprivation (to induce
of secretome eases its scale-up production, its storage, packaging and metabolic impairment). Another study used the human neuroblastoma
transportation, simplifies adjustment of specific dosages and avoids SH-SY5Y cell line subjected to scratch injury only (Kappy et al., 2018).
possible issues associated with cell infusion, for example MSC retention When ASC-derived secretome treatment was applied to scratch injured
in non-target organs after systemic infusion (Thäte et al., 2021) or neurons it reduced cell death, attenuated mitochondrial dysfunction and
vascular obstructions (Ge et al., 2014). decreased expression of the pro-inflammatory cytokine, IL-1β (Kappy
This review was conceived to first analyze in vitro and in vivo TBI et al., 2018). Studies on injured astrocytes showed that the secretome
studies exploring the effects of the secretome on clinically relevant improved wound healing responses (Torrente et al., 2014) by increasing
neurological and histopathological outcomes. To this end we conducted cell viability/proliferation and reversing morphological changes, as well
a comprehensive literature search to provide a narrative review of the as increasing polarity index and cell migration in a dose-dependent
therapeutic effects of secretome in TBI. Next, we discuss the central role manner (Baez-Jurado et al., 2018a). Among secretome-induced mech
of neuroinflammation in determining outcomes after injury, and we anisms of action, a clear benefit on mitochondrial function has been
analyze the immunomodulatory properties of the secretome as a key shown, with the reduction of free radicals (O2-) (Torrente et al., 2014),
mechanism in altering neuroinflammatory responses and providing the preservation of mitochondrial membrane potential (Baez-Jurado
neuroprotection. We end by discussing strategies to boost secretome et al., 2018a), the increased expression of mitochondrial antioxidant
activity, which will be required to increase the potential clinical use of enzymes (SOD2, GPX-1 and catalase) (Baez-Jurado et al., 2018a,
this novel cell-free based therapeutic strategy for TBI. 2018b), and the reduced expression of genes associated with fusion
(mfn1, mfn2) and fission (Fis1 and Drp1) (Baez-Jurado et al., 2019a).
2
The reduction of oxidative stress decreased DNA damage and nuclear 1.2. Neuroinflammation is a key contributor of outcome in TBI
fragmentation (Baez-Jurado et al., 2018a, 2018b), indicating a specific
effect of the secretome treatment in reducing apoptosis. Other mecha Neuroinflammation plays a crucial role in secondary injury pro
nisms of action included the modulation of inflammatory cytokines with gression, and its modulation may be crucial for optimal functional re
decreased IL-6, TNFα and GM-CSF expression, and increased expression covery. Cellular membrane disruption as a result of the primary
of IL-2 and IL-8 (Baez-Jurado et al., 2019a). Interestingly, secretome mechanical insult causes the release of damage associated molecular
treatment increased astrocytic production of Neuroglobin (Ngb). When patterns (DAMPs) and alarmins from the damaged meninges, glial lim
Ngb was silenced by siRNA the protective effects of the secretome on itans and brain parenchyma. These potent signals bind to pattern
mitochondrial injury in astrocytes was broadly attenuated (Baez-Jurado recognition receptors (PRRs) and cytokine receptors on CNS resident
et al., 2019a; Baez-Jurado et al., 2018b). glial cells that initiate a robust neuroinflammatory response by pro
In TBI models in rodents, secretome ameliorates neurological out ducing cytokines and chemokines, which coordinate the recruitment of
comes and improves functional recovery (table 2). Improvements in peripheral immune cells to sites of tissue damage. DAMPs interact with
sensorimotor deficits (Chang et al., 2013; Chuang et al., 2012; Tajiri receptors such as Toll-like receptors (TLRs), NOD-like receptors (NLRs),
et al., 2014; Xu et al., 2020), and cognitive function including spatial and scavenger receptors to detect endogenous danger molecules
learning and memory (Chang et al., 2013; Liu et al., 2020a; Tajiri et al., released from damaged or dying cells. DAMPs can also direct inflam
2014; Xu et al., 2020), in injured animals were shown by multiple matory signaling after TBI via activation of inflammasomes, intracel
behavioral tests. In addition, in a zebrafish TBI model, the secretome lular multiprotein complexes involved in the activation of caspase 1 and
reduced anxiety-like behaviors (Liu et al., 2020b). When assessing visual generation of mature IL-1β and IL-18. Inflammasomes can be assembled
function, Jha et al. showed that intravitreal injection of ASC-derived in multiple CNS populations, including microglia, astrocytes and neu
secretome in blast injured mice mitigated loss of visual acuity and pre rons, as well as brain infiltrating macrophages. In the CNS, the NLRP1
served morphological integrity of the retina (Jha et al., 2018, 2019, and NLRP3 inflammasomes appear to be the most relevant in injury and
2021). Furthermore, secretome treatment in TBI animals reduced chronic neurodegeneration. Increased levels of NLRP1, apoptosis-
vasogenic edema (Xu et al., 2020) and contusion volume (Chang et al., associated speck-like protein containing a caspase activation and
2013; Chuang et al., 2012; Tajiri et al., 2014), rescued neuro recruitment domain, and caspase 1 are found in the CSF of severely
degeneration (Tajiri et al., 2014), and apoptosis (Chang et al., 2013; Xu injured TBI patients and are associated with unfavorable neurological
et al., 2020), and induced neuronal proliferation and differentiation outcomes (Adamczak et al., 2014).
(Chang et al., 2013; Liu et al., 2020a). Secretome also has powerful
immunomodulatory properties, reducing the expression of pro-
1.3. Secretome mediated immunomodulation
inflammatory cytokines (e.g. IL-6 and TNF-α) and concomitantly
increasing anti-inflammatory ones (e.g. TGF-β) in injured brain tissue,
MSCs alter immune cell activation and functions both in vitro and in
with a shift of microglia/macrophage activation toward protective
vivo, primarily via paracrine mechanisms. Here, we discuss the evidence
phenotypes (decrease of Iba1+/NOS2+ and increase of Iba1+/Arg1+
of the secretome induced immunomodulatory action on peripheral and
cells) (Xu et al., 2020). Surprisingly, very little is known about the ef
central innate and adaptive immune cells following TBI, and we focus on
fects of the secretome on systemic inflammation after TBI.
the immune factors mediating their protective actions.
Table 1
In vitro studies assessing secretome efficacy in TBI models.
Reference MSC Secretome TBI model Delivery protocol Outcomes
source production
Torrente, 2014 Human 48h • Cell line: Human astrocytes T98G • Immediately after At 12, 24 and 48h:
ASC Passage 3-4 • Injury: Scratch injury + Glucose injury ↑ wound closure
deprivation ↓ ROS production
↑ cell proliferation
Kappy, 2018 Human 48-72h • Cell line: Human neuroblastoma SH- • 2% of culture At 24h:
ASC Passage 1-3 SY5Y medium ↑ cell survival
• Injury: Scratch injury • Immediately after ↓ inflammatory markers (IL-1β)
injury ↓ apoptosis
Baez-Jurado, Human 48h • Cell line: Human astrocytes T98G • 15%of culture At 30h:
2018a ASC Passage 3-5 • Injury: Scratch injury + Glucose medium ↑ cell survival
deprivation • Immediately after ↑ mitochondrial membrane potential
injury ↑ antioxidant proteins (SOD2, GPX-1)
↑ Ngb
Baez-Jurado, Human 48h • Cell line: Human astrocytes T98G or • 2%of culture medium At 24h:
2018b ASC Passage 3-5 T98G-siRNA Ngb • Immediately after ↑ cell survival
injury ↑ cell proliferation
• Injury: Scratch injury + Glucose ↓ ROS production
deprivation ↑ antioxidant proteins (SOD2, GPX-1, catalase)
↑ Ngb
When Ngb is silenced secretome efficacy is reduced

Baez-Jurado, Human 48h • Cell line: Human astrocytes T98G or • 2%of culture medium At 24h:
2019 ASC Passage 3-5 T98G-siRNA Ngb • Immediately after ↓ pro-inflammatory markers (IL-6, TNFα, GM-CSF)
injury ↑ IL-2, IL-8
• Injury: Scratch injury + Glucose ↑ survival cascades (AKT/pAKT, ERK/pERK)
deprivation ↓ intracellular Ca2+
↑ mitochondrial health (modulation of fusion and
fission genes)
When Ngb is silenced secretome efficacy is reduced
Abbreviations: ASC = adipose stem cells, ROS = reactive oxygen species, Ngb = Neuroglobin, GM-CSF = Granulocyte-Macrophage Colony-Stimulating Factor
3
Table 2
In vivo studies assessing secretome efficacy in TBI models.
Reference MSC Secretome Pre- Concentration TBI model Delivery Functional outcomes Histological Immune cell
source production conditioning protocol outcomes modulation
Chuang Human 24h // 25x Adult male • 500 μg At 3 days: At 3 days: //

TJ, BM 3kDa cutoff SD rats, FPI • IV ↑ motor function (limb ↓ infarction
2012 • post TBI strength) ↓ apoptosis
↑ neuronal
density
↑ VEGF in cortex
Chang CP, Human 24h Normoxia vs 25x Adult male • 100 μg/kg At 4 days: At 4 days: //
2013 BM hypoxia (0.5 3kDa cutoff SD rats, FPI • IV ↑ motor function (limb ↓ contusion
O2) • Daily x 3d strength) volume
↑ cognitive function ↓ neuronal
(PA) apoptosis
↑ neurogenesis
Tajiri N, Human 24h // 1x Adult and • 500 μl At 1,3 and 7 days in At 11 days in //
2014 ASC aged male • IV young but not aged young but not
F344 rats, • 3h rats: aged rats:
CCI ↑ motor function ↓ contusion
(EBST, forelimb volume
akinesia, paw grasp) ↓ neuronal
At 11 days: apoptosis
↑ cognitive function
(RAWM)
Jha KA, Human 24h TNFα + INFγ 20x Adult male • 1 μl At 4 weeks: At 4 weeks: At 4 weeks in
2018 ASC C57BL/6 • Intravitreal ↑ visual function ↑ morphological the retina:
mice (optokinetic reflex) integrity in the ↓ reactive
blast injury retina gliosis
(GFAP)
↓ pro-infl
markers
(CD86, IL1β,
CD68)
Jha KA, Human 24h TNFα + INFγ 20x Adult male • 1 μl At 4 weeks: // At 4 weeks in
2019 ASC 3kDa cutoff C57BL/6 • Intravitreal ↑ visual function the retina:
siRNA to block mice (optokinetic reflex, ↓ reactive
TGS-6 blast injury electroretinogram) gliosis
(GFAP)
The effect is lost when ↓ pro-infl
TSG-6 is blocked markers
The effect is
lost when
TSG-6 is
blocked
Liu XY, Human 24h Exposure to 100 kDa Adult male • 3 μl At 4 weeks: At 7 and 4 weeks: //
2020 UC tissue extract cutoff SD rats • Dentate gyrus ↑ cognitive function ↑ neurogenesis
from TBI rats FPI of (MWM) ↓ apoptosis
for 24h hippocampus
• post TBI Effect enhanced with Effects enhanced
preconditioned with
secretome preconditioned
secretome
Xu C, Human 24h hypoxia (5% 5 kDa cutoff Adult male • 100 μl At 4 weeks: ↓ vasogenic Microglia:
2020 ASC O2) SD rats • IV ↑ sensorimotor edema (MRI) ↓ Iba1+/
CCI • post TBI than function (mNSS) ↓ apoptosis iNOS+
daily up to 7d ↑ cognitive function ↑ Iba1+/
(MWM) Arg1+
Cytokines:
↓ IL-6, TNFα
↑TGFβ, TSG-
6
Liu XY, Human 72h // Zebrafish, • 4 μl At 6 and 24h: ↓ reactive
2020 UC pulsed high- • retro-orbitally ↑ locomotor activity gliosis
intensity • 30 min post ↓ anxiety behavior (GFAP)
focused injury (novel tank test)
ultrasound
Jha KA, Human 24h TNFα + INFγ 20x Adult male • 1 μl At 4 weeks: At 4 weeks in the At 4 weeks in
2021 ASC 3kDa cutoff C57BL/6 • Intravitreal ↑ visual function retina: the retina:
mice (electroretinogram) ↓ free glutamate ↓ reactive
blast injury ↑ GS and GLAST gliosis
↑ AQP4 (GFAP)
Abbreviations: ASC = adipose stem cells, BM = bone marrow, AQP = aquaporin-4, CCI = controlled cortical impact, EBST = elevated body swing test, FPI = fluid
percussion injury, GLAST = glutamate-aspartate transporter, GS = glutamine synthetase, glutamate-aspartate transporter, RAWM = radial arm water maze, TSG-6 =
TNF-stimulated gene 6 protein, PA = passive avoidance
4
1.3.1. Secretome effects on the innate immune responses following TBI modulated by BM-MSCs via the soluble form of TNFα receptor 1
Neutrophils are the most abundant cell population in circulation (sTNFR1; Figure 1). Rats subjected to a lipopolysaccharides (LPS)
after TBI (Liao et al., 2013). They are the first peripheral immune cells to endotoxemia model and transplanted with BM-MSCs had decreased
be recruited to the damaged meninges and brain parenchyma in circulating pro-inflammatory cytokines (e.g. TNFα, IFNγ, IL-6) and
response to chemokines gradients (e.g. IL-8/CXCL8) produced immedi reduced infiltration of neutrophils and macrophages in three vital or
ately after TBI (Helmy et al., 2011). Vascular endothelium cell adhesion gans (lungs, kidney, and liver) (Yagi et al., 2010). These beneficial ef
molecules, such as E-selectin and intercellular adhesion molecule 1 fects were partially abrogated when MSCs were co-delivered with a
(ICAM1) (Pleines et al., 1998; Whalen et al., 1998), are also upregulated neutralizing antibody against sTNFR1 (Yagi et al., 2010).
after injury and facilitate neutrophil trafficking into damaged brain In the context of TBI, MSCs have been shown to reduce neutrophil
parenchyma. Neutrophils are recruited within hours of the brain injury infiltration in contused tissue (Watanabe et al., 2013; Zhang et al.,
and predominate within the lesion microenvironment during the first 2013), with an associated decrease of pro-inflammatory cytokines (e.g.
few days (Clark et al., 1994; Soares et al., 1995). They are then greatly IL-1β, IL-17, TNFα, IFNγ) and an increase anti-inflammatory cytokines
diminish in numbers from 5-7 days post-injury (Clark et al., 1994; Soares (e.g. IL-10, TGF-β1) expression. This phenotypic switch correlated with
et al., 1995). Their functional role is in the containment of the injury improved sensorimotor function in TBI rats (Zhang et al., 2013). These
lesion and in removal of cellular debris (Liu et al., 2018). However, they effects were associated with an increased expression of TNF stimulated
can also compound brain injury by direct toxic effects of matrix metal gene-6 (TSG-6 in the MSC treated TBI group with a concomitant
loproteinases (MMPs), reactive oxygen species (ROS), and TNFα downregulation of the NF-κB pathway, suggesting a role for TSG-6 in the
(Nguyen et al., 2007), as well as by increasing vascular permeability and beneficial effects of MSC treatment (Zhang et al., 2013). When TSG-6
edema formation (Liu et al., 2018). Experimental studies using anti-GR1 alone was administered to TBI mice, neutrophil infiltration was mark
antibody to deplete neutrophils in a controlled cortical impact (CCI) edly decreased in the contusion area to similar levels as observed in the
model in mice resulted in reduced edema, neuronal apoptosis, and brain MSC treated TBI mice (Watanabe et al., 2013). Further, MSCs and TSG-6
tissue loss acutely after injury (Kenne et al., 2012). Furthermore, treatments also protected against post-traumatic BBB damage as
disruption of neutrophil infiltration or function using genetic deletion of demonstrated by reduced evans blue dye extravasation and a concomi
CXCR2 and neutrophil elastase, respectively, reduced post-traumatic tant reduction in MMP-9 activity (Watanabe et al., 2013).
edema and neuronal cell death, but failed to improve neurological Another MSC-released factor involved in BBB preservation is the
outcomes after CCI (Semple et al., 2015; Semple et al., 2010). tissue inhibitor of matrix metalloproteinase-3 (TIMP3, Figure 1). TBI
Neutrophils can also contribute to blood brain barrier (BBB) damage. mice that received two intravenous MSC administrations at 2 and 24
Neutrophil-derived MMPs are induced within hours of TBI and degrade hours post-injury had increased serum TIMP3 concentration and an
structures of the neurovascular unit leading to BBB dysregulation overall decrease in BBB permeability (Menge et al., 2012). Notably,
(Grossetete et al., 2009; Hayashi et al., 2009). There is a unique tem siRNA mediated down-regulation of TIMP3 expression in MSCs abro
poral expression pattern of MMPs in extracellular brain fluid from severe gated their protective actions on BBB stability after TBI, while admin
TBI patients, with MMP-8 and MMP-9 appearing first, followed by MMP- istration of recombinant TIMP3 alone recapitulated their protective
2 and MMP-3, and finally MMP-7 (Roberts et al., 2013). Notably, MMP-8 actions and resulted in reduced neutrophil infiltration and preserved
is associated with increased ICP in severely injured patients and worse BBB integrity after TBI (Menge et al., 2012).
overall outcomes. Following CCI in mice there is increased MMP-9 There is a close relationship between neutrophil apoptosis and ROS
expression in injured brain through one week post-injury. MMP-9 pro production. In vitro studies have shown that BM-MSCs decrease ROS
motes BBB breakdown and vasogenic edema acutely after TBI (Shige production and apoptosis in both naive and IL-8-activated neutrophils
mori et al., 2006), while genetic ablation of MMP-9 is neuroprotective, via mechanisms that are independent of cell-cell contact (Raffaghello
reducing the brain lesion and improving TBI-induced motor function et al., 2008) and dependent on IL-6 (Figure 1). In fact, pre-treatment of
deficits when compared to wild-type TBI control mice (Wang et al., the secretome with a neutralizing anti-IL-6 monoclonal antibody abol
2000). Neutrophils are also a major source of free radicals, including ished the protective actions, while supplementation of MSCs with re
ROS, nitrous oxide (NO), and NADPH oxidase (NOXs) enzymes (Liao combinant IL-6 prevented apoptosis of neutrophils (Raffaghello et al.,
et al., 2013). Free radicals induce direct oxidative damage, which can 2008). Of note, these findings were replicated with MSCs from peri
result in downregulation of claudin-5 and occludin in endothelium odontal ligament (Wang et al., 2017), indicating that IL-6 in the secre
leading to BBB degradation (Pun et al., 2009). tome is a critical modulator of neutrophil apoptosis and ROS production.
Neutrophil recruitment to inflamed tissue has been shown to be Stromal cell-derived factor-1 (SDF-1, Figure 1) released by MSCs has
Fig. 1. MSC secreted factors that alter im

mune activity in neutrophils. MSCs can 1)
reduce neutrophil tissue infiltration via the
release of the soluble form of TNFα receptor
1 (sTNFR1) or TNF stimulated gene-6 (TSG-
6); 2) reduce blood brain barrier (BBB)
permeability after injury through TSG-6 and
tissue inhibitor of matrix metalloproteinase-
3 (TIMP3); 3) reduce neutrophil apoptosis
and reactive oxygen species (ROS) release
via interleukin-6 (IL-6); 4) stimulate the
phagocytic activity of neutrophils via stro
mal cell-derived factor-1 (SDF-1). Figure
created in BioRender.com
5
been shown to regulate neutrophil phagocytic function. In mice that monocytes in TBI is less clear. Two research groups used CX3CR1
were subjected to a polymicrobial sepsis induced by cecal ligation and knockout (CX3CR1-/-) mice to disrupt CX3CL1 chemokine signaling and
puncture and treated 2 hours later via intravenous administration of non-classical monocyte trafficking to the injured brain, and both iden
MSCs, there was an increase in survival rates compared to non-treated tified a time-dependent role for CX3CL1/CX3CR1 signaling after TBI
counterparts (from 10% to 70% at 7 days post-sepsis) (Kwon et al., (Febinger et al., 2015; Zanier et al., 2016). During the acute phase after
2020). Notably, the protective actions of MSCs in septic mice were lost CCI (up to 15 days post-injury), motor deficits and neuronal cell death
when SDF-1 production in MSCs was silenced by shRNA (Kwon et al., were greater in wild-type TBI mice than in CX3CR1− /− TBI mice.
2020). Additional in vitro studies demonstrated that MSC-released SDF-1 However, during the chronic phase at 30 days post-injury, CX3CR1− /−
increased neutrophil phagocytosis of bacteria. These protective phago TBI mice exhibited greater cognitive and motor dysfunction and
cytic responses in neutrophils were lost when SDF-1/CXCR4 signaling increased neuronal death than wild-type TBI mice. The protective and
was blocked in MSCs, either by shRNA knockdown of SDF-1, or by deleterious effects of CX3CR1 were associated with changes in macro
culturing MSC-treated neutrophils with a CXCR4 antagonist (Kwon phage/microglia activation status (Febinger et al., 2015; Zanier et al.,
et al., 2020). Respiratory burst in neutrophils and their phagocytic re 2016). Notably, during the acute phase post-injury CX3CR1− /− TBI mice
sponses are critical for acute host defense and removal of cellular debris expressed anti-inflammatory macrophage/microglia markers (e.g. Ym1,
after TBI. Moreover, TBI patients are highly vulnerable to infections in CD206, and TGFβ), whereas at later time points the CX3CR1− /− TBI
the acute post-injury period (Sharma et al., 2019), and thus faster mice expressed greater pro-inflammatory activation markers (e.g.
elimination of pathogenic bacteria by secretome stimulated neutrophils NOS2, Marco, and CD68). These studies indicate that while early
could lead to improved recovery trajectories in severely TBI patients CX3CR1 signaling may have detrimental effects after TBI, this signaling
with secondary infectious complications. is required to prevent long-term neuroinflammation and neurological
Monocytes are circulating bone marrow–derived leukocytes that impairments.
differentiate into macrophages or dendritic cells (DCs) after invasion of Microglia are the resident innate immune cells of the CNS, act as first
injured tissue. They perform critical innate immune functions, such as line of defense and have critical functions in neuroinflammatory re
phagocytosis, cytokine/chemokine release, antigen presentation, im sponses to acute brain injury. Microglia share a common origin with
mune modulation, and tissue repair. Following TBI, monocytes enter the monocytes, deriving from primitive erythromyeloid progenitors during
perivascular spaces and brain parenchyma within 1-2 days, differentiate embryogenesis and express common markers such as CD11b, IBA1,
into macrophages, and they can reside in brain for several weeks CD45, CD68, F4/80, CX3CR1 (Jurga et al., 2020). After TBI, microglia
(Beschorner et al., 2002). Blood-derived monocytes are recruited to the rapidly activate, contract their processes, change morphology from
injured brain in response to local chemokine gradients (e.g. CCL2, highly ramified to hypertrophic/bushy to amoeboid shape so that it
CCL5), which are found in the CSF or plasma of TBI patients (Lumpkins becomes difficult to morphologically distinguish them from infiltrated
et al., 2008; Semple et al., 2010). Once in the brain, they differentiate macrophages. Microglia can acquire a classical/inflammatory or a non-
into macrophage subpopulations distinguished by relative cell-surface classical/anti-inflammatory phenotypes (Loane and Kumar, 2016),
expression of the CCR2 and CX3CR1 (classical/inflammatory mono although it is clear that, due to the multifaceted inflammatory milieu in
cytes = CD11b+CD45hiCCR2+Ly6Chi; non-classical monocytes = the TBI-injured brain, it is not possible to draw a binary categorization
CD11b+CD45hiCX3CR1+ (Russo et al., 2018)). with evidence of mixed phenotypes (Morganti et al., 2016).
Inflammatory monocytes may play a pathogenic role after TBI To study the contribute of microglia after TBI, depletion studies have
because reducing CCR2+ monocyte recruitment decreases lesion vol been pursued. In particular microglia require CSF1R signaling for their
ume and improves neurological recovery in experimental models of TBI. survival and pharmacological approaches based on CSFR1 inhibition
Using a CCL2 knockout (CCL2-/-) model to disrupt CCL2/CCR2 (using Plexxikon drug –PLX5622) are routinely performed, allowing a
signaling, Semple et al. (2010), demonstrated that CCL2-/- mice sub >95% depletion of all microglia in the CNS with 7 days of treatment
jected to TBI by closed head injury (CHI) had reduced cytokine (Elmore et al., 2014). This method allows a “reset” of microglia acti
expression acutely after injury, but there was no overall effect on lesion vation, since, after the cessation of CSF1R inhibition, the spared/sur
volume. However, at later time points there was a reduction in lesion viving microglia can ex novo repopulate the brain parenchyma (Elmore
volume, macrophage/microglial activation and reactive astrocytes in et al., 2014). Using this depletion strategy and removing microglia at 1
the thalamus of CCL2-/- mice compared to wild type TBI mice (Semple month after CCI, it has been demonstrated that the repopulated micro
et al., 2010). This delayed neuroinflammatory change corresponded glia displayed a ramified morphology similar to non-injured animals,
with improved long-term motor function recovery in TBI mice (Semple while vehicle-treated TBI animals showed persistent hypertrophic
et al., 2010). Similar results were obtained in CCR2-/- mice subjected to microglia (Henry et al., 2020). This microglial reset, led to improvement
either fluid percussion injury (FPI) or CCI models of experimental TBI of sensorimotor and cognitive function, a reduced tissue loss 3 months
(Gyoneva et al., 2015; Hsieh et al., 2014). When compared with levels in after injury and a modulation of cortical gene expression with reduced
wild-type TBI mice, CCR2-/- TBI mice had approximately 80-90% NLRP3 inflammasome, oxidative stress and apoptotic related-gene
reduced numbers of infiltrating brain macrophages in the lesion site activation (Henry et al., 2020). Depleting microglia before TBI is also
acutely after injury, which coincided with preserved neuronal density in beneficial and reverses neurodegenerative/damage-related neuronal
the CA1-CA3 regions of the hippocampus. Furthermore, CCR2-/- TBI genes associated with a preserved morphological neuronal complexity 7
mice had improved long-term motor and cognitive performance when days after injury and functional recovery at 1 month (Witcher et al.,
compared to wild-type TBI mice (Hsieh et al., 2014). These findings 2021). These data indicate that microglia contribute to neurodegener
were supported by pharmacological studies that targeted CCL2/CCR2 ative processes and that strategies aimed at reprogramming their acti
signaling using a CCR2 antagonist, CCX872, in a CCI model in wild-type vation after TBI could contribute to the resolution of the damage.
C57BL/6 mice. When compared to levels in vehicle-treated TBI mice, MSCs can alter macrophage activation (Carty et al., 2017), and their
prophylactic administration of CCX872 reduced macrophage accumu secretome can inhibit monocyte differentiation toward mature DCs
lation in the injured brain and altered pro- and anti-inflammatory (Ortiz-Virumbrales et al., 2020; Spaggiari et al., 2009) and switch the
cytokine levels, as well as markers of NADPH oxidase and oxidative differentiation of classical/inflammatory monocytes into non-classical/
damage (Morganti et al., 2015). These inflammatory and oxidative regulatory ones (Magatti et al., 2017). In the context of TBI, anti-
changes were associated with less severe hippocampal-dependent inflammatory actions of MSC therapy (Zanier et al., 2014; Zanier
cognitive impairments after TBI in a radial arm water maze task at 30 et al., 2011; Zhang et al., 2013), secretome (Jha et al., 2018; Ooi et al.,
days post-TBI (Morganti et al., 2015). 2015; Xu et al., 2020) or MSC-EVs (Chen et al., 2020) on microglia/
In contrast to inflammatory monocytes, the role of non-classical macrophages response during secondary injury have been described.
6
Here, we discuss MSC-released identified factors that alter myeloid cell inflammatory phenotype in macrophages that lack IL-6Rα, which have
activation and function (Figure 2). persistent up-regulation of TNFα and no IL-10 expression (Philipp et al.,
The prostaglandin, PGE2, plays a crucial role in the induction of an 2018). Collectively, IL-10 and IL-6 signaling appears to significantly
anti-inflammatory phenotype in myeloid cells (Figure 2). PGE2 release contribute to MSC-induced immunomodulation. Another MSC-released
from MSCs varies depending on the cell source and experimental pro cytokine that has a role in immunomodulation is TGF-β (Figure 2). In
tocol, with laboratories showing its secretion in basal condition from vitro, secretome treatment inhibited microglial activation following LPS-
ASCs (Yañez et al., 2010), BM-MSCs (Kota et al., 2017; Spaggiari et al., stimulation resulting in decreased pro-inflammatory cytokine expres
2009; Yañez et al., 2010) and AMSCs (Magatti et al., 2017), while other sion (e.g. TNFα, IL-1β, IL-6, NOS2), an induction of markers of non-
groups showing PGE2 release only after an inflammatory stimulus to classical activation (e.g. CX3CR1), and enhanced microglial phagocy
ASCs (Ortiz-Virumbrales et al., 2020) or BM-MSCs (Németh et al., 2009). tosis (Noh et al., 2016). When TGF-βR signaling was blocked using
In any case, after PGE2 blockade, MSCs or their secretome are no longer neutralizing antibodies or siRNA knockdown the anti-inflammatory
able to inhibit the maturation of monocyte-derived DCs (Ortiz-Virum activity of the secretome in microglia was lost (Noh et al., 2016).
brales et al., 2020; Spaggiari et al., 2009) or induce an anti- MSC-secreted TSG-6 also regulates myeloid cell phenotype and
inflammatory phenotype (Magatti et al., 2017; Ylöstalo et al., 2012), function (Figure 2). Intravenous injection of MSCs into naïve mice
highlighting an important regulatory role of MSC-derived prostanoids induce a distinct population of MHC class II+B220+CD11b+ monocytes/
for immunomodulation. In a model of sepsis, it has been demonstrated macrophages in the lungs that upregulated IL-10 (Ko et al., 2016). This
that intravenously delivered MSCs reduce mortality in mice and modify population of circulating monocytes suppressed T-cell proliferation and
lung monocytes/macrophages activation with increase IL-10 production induced tolerance after corneal allograft (Ko et al., 2016). Importantly,
via the release of PGE2 (Németh et al., 2009). In the context of TBI, BM- the protective effects of this unique anti-inflammatory population were
MSC-released PGE2 modulates microglia activation in the injured cor completely lost when MSCs were transfected with siRNA targeting TSG-
tex, such that suppression of COX2 impaired the ability of BM-MSCs to 6 (Ko et al., 2016). Similarly, inhibitory experiments targeting TSG-6 in
inhibit post-traumatic neuroinflammatory responses (Kota et al., 2017). MSCs demonstrate that it can alter microglial activation because TSG-6
There appears to be a donor-dependent variability in the expression of inhibition in MSCs prevented their anti-inflammatory actions in an LPS-
COX2 and PGE2 because their levels directly correlated with MSC model of microglial activation (Jha et al., 2019; Liu et al., 2014). To
immunosuppressive activity in vitro (as evaluated by their ability to confirm TGS-6’s action, recombinant TGS-6 was directly added to LPS-
suppress TNFα and IFNγ in activated splenocytes). Following TBI, MSCs stimulated microglia, which resulted in reduced pro-inflammatory
with higher levels of COX2 expression showed greater efficacy at gene expression, thereby mirroring the anti-inflammatory gene expres
attenuating post-traumatic neuroinflammation, indicating that PGE2 sion pattern induced by MSC treatment (Liu et al., 2014). Specifically,
levels may be a predictive marker of MSC potency (Kota et al., 2017). TSG-6 attenuated NF-κB signaling and increased phosphorylation of
Among MSC-secreted cytokines, IL-10 and IL-6 are critical regulators p38, JNK and ERK, which resulted in an anti-inflammatory activation
of macrophage polarization (Figure 2). The BM-MSC-derived secretome state that was CD44 dependent (Liu et al., 2014).
contains high levels of IL-10, which alters macrophage activation by Glial cell-derived neurotrophic factor (GDNF) is another factor that
upregulating anti-inflammatory CD206+ cells (Dang et al., 2022). can modulate microglial activation (Figure 2). BM-MSCs increase pro
Notably, neutralizing anti-IL-10 antibodies prevented the protective and duction of GDNF following TNFα stimulation (Zhong et al., 2020). In LPS
anti-inflammatory effects of the secretome in these studies (Dang et al., stimulated microglia, BM-MSCs or GDNF alone decreased pro-
2022). In addition, the MSC inhibitory effects on the differentiation of inflammatory cytokines (e.g. TNFα, IL-1β) and increased anti-
monocytes to DCs is mediated by IL-6, whereby IL-6 transform mono inflammatory IL-10, and this microglial phenotypic shift was pre
cytes toward an IL-10 producing anti-inflammatory activation state with vented when GDNF was knocked down by siRNA (Zhong et al., 2020). In
decreased expression of co-stimulatory CD14 molecule (Deng et al., vivo, MSC infusion increased the expression of GDNF in the contused
2016; Djouad et al., 2007). Interestingly, MSCs do not induce the anti- tissue and was associated with better neurological outcomes after
Fig. 2. MSC secreted factors with immune

activity in myeloid cells. MSCs can 1) inhibit
the differentiation of monocytes to dendritic
cells through the release miR-21-5p con
tained in extracellular vesicles (EV), prosta
glandin E2 (PGE2), interleukin – 6 (IL-6); 2)
induce a non-classical/anti-inflammatory
macrophage activation through miR-21-5p,
PGE2, IL-6, IL-10, TNF stimulated gene-6
(TSG-6); 3) induce a non-classical/anti-
inflammatory activation of LPS-stimulated
microglia through TSG-6G, transforming
Growth Factor-β (TGF-β) and glial cell-
derived neurotrophic factor (GDNF). Figure
created in BioRender.com
7
experimental TBI (Shen et al., 2016). However, whether GDNF acts via neuroprotection (Hauben et al., 2000; Hofstetter et al., 2003). The
the modulation of immune cells during TBI is still not clear. protection may be due to CD4+ T cell release of IL-4 within the local
MSC-derived extracellular vesicles (MSC-EVs) can act as a shuttle injury microenvironment that acts via neuronal IL-4 receptors to in
mechanism for immunomodulatory factors, including microRNAs (miR) crease neurotrophin signaling that promotes recovery in injured axons
that regulate gene expression through mRNA silencing. For example, (Walsh et al., 2015). The discrepancy in experimental literature
BM-derived MSC-EVs impair maturation of monocyte-derived DCs in regarding pathogenic or protective autoimmunity following TBI may be
vitro, decrease activation markers CD83, CD38 and CD80, reduce the due to differing T-lymphocyte populations contributing to specific
release of the pro-inflammatory cytokines such as IL-6 and IL-12p70, detrimental and beneficial injury responses. Further mechanistic studies
and increase the production of anti-inflammatory cytokine TGF-β (Reis will be required to define cellular and molecular pathways for both.
et al., 2018). MSC-EVs have also been shown to reduce CCR7 expression The MSC-mediated modulation of adaptive immune response is
in DCs following LPS stimulation, thereby preventing migration along mainly focused on T cells. In vitro studies have identified different me
the CCR7-CCL21 signaling axis that is required for DC homing to the diators contained in the secretome that alter T cell proliferation and
lymph nodes for antigen presentation. Interestingly, miR-21-5p, is also activation. The enzyme indoleamine 2,3-dioxygenase (IDO, Figure 3) is
highly enriched in MSC-EVs, where it has been shown to target CCR7 released from MSC and inhibits T cell proliferation. IDO is a rate-limiting
mRNA for degradation (Figure 2). Reis et al. (2018), transfected DCs enzyme that catalyzes the first step of tryptophan (TRP) degradation
with miR-21-5p mimics and observed reduced DC migration along the through the kynurenine pathway. TRP is essential for T cell prolifera
CCR7-CCL21 signaling axis (Reis et al., 2018). MSC-EV shuttled miR-21- tion, thus its loss leads to T cell cycle arrest and apoptosis (Lee et al.,
5p has also been demonstrated to have an immunomodulatory role in 2002). Moreover the TRP catabolites L-kynurenine (KYN), 3-hydroxyky
macrophages. LPS stimulated RAW264.7 macrophages that were treated nurenine (3HK) and 3-hydroxyanthranilic acid (3-HAA) have been
with MSC-EVs had reduced pro-inflammatory (e.g. NOS2, IL-1β, IL-6 and shown to inhibit T cell proliferation in vitro in a concentration-
TNFα) and increased anti-inflammatory (e.g. Arg1, CD206 and IL-10) dependent manner (Dai and Zhu, 2009). There is an intrinsic donor-
marker expression. These phenotypic transitions were abolished when related variability in the amount of IDO production. Higher levels of
MSCs were cultured with miR-21-5p antagomirs, and were restored IDO are associated with greater inhibition of T cell proliferation and IDO
when miR-21-5p mimics were directly transfected into macrophages blockage results in a complete loss of MSC ability to suppress T cell
(Shen and He, 2021). proliferation (François et al., 2012; Torres Crigna et al., 2020). More
over, it has been shown that KYNA activates the aryl hydrocarbon re
1.3.2. Secretome and adaptive immune responses following TBI ceptor (AhR) promoting its translocation into the nucleus, thereby
The release of brain antigens into the systemic circulation after TBI directly promoting TSG-6 expression by binding to its promoter (Wang
may activate adaptive immune response, including humoral immunity, et al., 2018), boosting the immunomodulatory effects described in the
mediated by B-lymphocytes producing antibodies, and cell-mediated above paragraphs.
immunity, directed by effector T-lymphocytes (Needham et al., 2019). Inhibitory actions on T cell proliferation can also be mediated by
However, clinical and experimental studies provide conflicting evidence prostaglandins (PGs, Figure 3), which are largely secreted by MSCs
of both protective and pathogenic autoimmunity mediated by specific (Rossi et al., 2012). Specifically, when MSCs are cultured in the presence
CNS antigens. Autoreactive T cell specific to CNS antigens have been of cyclooxygenase inhibitors, which dramatically reduce the secretion of
isolated from the blood of TBI patients (Cox et al., 2006), and TBI can PGs, the secretome had greatly reduced anti-proliferative capacity on T
induce a cell-mediated immune response with T cells being recruited to cells in culture (Ghannam et al., 2010; Rossi et al., 2012). Programmed
the CNS independent of their specificity (Hirschberg et al., 1998). death-1 (PD-1) receptor, expressed on the cell surface of activated T and
The recruitment of T cells into the traumatically injured brain occurs B cells, is an important lymphocyte regulator since its interaction with
soon after monocyte infiltration (Holmin et al., 1998), and through the ligands PD-L1 and PD-L2 provides an inhibitory signal (Parry et al.,
upregulation of cell adhesion molecules and in response to local che 2005). MSCs can express these ligands on their surface and are able to
mokines, such as CCL5 (Helmy et al., 2011). Following TBI there is a inhibit T cell proliferation in a contact dependent manner (Augello et al.,
massive release of CNS-specific proteins, including glial fibrillary acidic 2005; Wang et al., 2015). More recently it has also been shown that both
protein (GFAP), myelin basic protein (MBP), myelin oligodendrocytes resting and licensed (by INFγ and TNFα) MSCs could release soluble (s)
glycoprotein (MOG), and myelin-associated glycopeptide (MAG) forms of PD-1 ligands (sPD-L1 and sPD-L2, Figure 3), providing evidence
(Needham et al., 2021; Needham et al., 2019; Zhang et al., 2014), both for a contact-independent mechanism (Davies et al., 2017). Specifically,
directly into the circulation and to the cervical lymph nodes via the transwell co-culture of MSCs with T cells results in T cells suppression
glymphatic and meningeal lymphatic systems (Plog et al., 2015). CNS (as revealed by the decreased expression of CD25) (Davies et al., 2017).
proteins can be phagocytosed by professional antigen-presenting cells Addition of an anti-PD-1 blocking antibody reversed the inhibition of
which results in production of self-reactive T-cells. Once activated, T- CD25 expression, while blockade of single ligands separately results in a
cells upregulate the necessary surface molecules to migrate across the partial effect, indicating that sPD-L1 and sPD-L2 play a synergistic role
BBB, and antigen-specific CD8+ T-cells have been demonstrated to in exerting T cell suppression. Mechanistically, both ligands suppress the
migrate into the CNS and co-localize to their cognate antigen (Ling et al., T cell release of effector cytokine IL-2, blocking the auto-paracrine
2006). In the CNS, autoreactive T-cells are typically considered to be positive feedback loop on T cell activation and leading to cell toler
harmful, such as in the case of Multiple Sclerosis. Recent experimental ance/anergy and death (Davies et al., 2017). Importantly, TBI results in
studies demonstrated that Th17-drive CD8+ T-cell responses instigated an acute transient expression of PD-L1 in reactive astrocytes and inhi
neurodegeneration by stimulating granzyme B/perforin effector path bition of PD-L1 signaling aggravates motor function deficits, increases
ways, which contribute to continual neuronal damage, myelin pathol lesion volume and is associated with higher rates of T cell infiltration
ogy, and neurological dysfunction for several months after severe CCI in into brain parenchyma (Gao et al., 2022). Therefore, PD-L1 could have
mice (Daglas et al., 2019). However, conversely, autoreactive T cells an important role as gatekeeper to control injury-related neuroimmune
specific to CNS antigen have also been shown to protect injured axons and neuroinflammatory responses. Its presence in the secretome may
following damage (Moalem et al., 1999). Passive transfer of activated therefore regulate neuroimmune signaling and prevent chronic neuro
MBP-reactive T cells when delivered to rats attenuated secondary inflammation after TBI.
degeneration of retinal ganglion cells following optic nerve crush injury
(Moalem et al., 1999). Additional studies of active immunization with 1.4. Immune system and aging: challenges for secretome therapy in TBI
CNS-specific myelin products, MBP or MOG, in models of traumatic
spinal cord and brain injury also demonstrated significant Aging is characterized by a gradual loss of brain volume associated
8
Fig. 3. MSC secreted factors with immune activity on T cells. MSCs secrete indoleamine 2,3-dioxygenase (IDO) that can reduce T cell proliferation by degrading
tryptophan (TRP) (amino acid essential for T cell proliferation) through the kynurenine pathway. TRP catabolites L-kynurenine (KYN), 3-hydroxykynurenine (3HK)
and 3-hydroxyanthranilic acid (3-HAA) and MSC-secreted prostaglandin E2 (PGE2) have direct inhibitory action on T cell proliferation. MSCs can also release soluble
(s) PD-L1 and sPD-L2 that induce T cell anergy and apoptosis. Figure created in BioRender.com
with cognitive and physical decline. Adverse biological effects associ bioactive factors reaching the brain, and/or by an immunomodulatory
ated with aging include accumulation of DNA damage and misfolded effect on immune cells which contribute to a protective/regenerative
proteins, oxidative stress, cellular senescence and impaired debris brain microenvironment when infiltrating the injured tissue. The
clearance. Old age increases microglial senescence and secondary neu observation that secretome treatment is protective in the young, but not
roinflammation, worsens white matter damage and greatly impairs in the aged TBI animals, could be explained by the reduced ability of the
neurological outcomes after experimental TBI (Moro et al., 2022; Ritzel aged brain to respond to trophic/regenerative stimuli, and/or by a
et al., 2019). These effects are associated with impaired acute systemic dysregulated immune response in the aged brain that is less responsive
immune function and parallel findings in patients showing significant to secretome immunomodulatory activity.
lower monocyte and lymphocyte counts in aged (>70 years-old) There are age-related immunomodulatory responsiveness to MSC
compared to young (<45 years-old) TBI patients (Moro et al., 2022). from human peripheral blood mononuclear cells (PBMCs) in healthy
All these factors indicate a reduced immunological competence in the donors (Guan et al., 2019). For example, PBMCs from young donors (26-
elderly that can impede the effectiveness of immunomodulatory treat 30 years-old) had suppressed proliferation and a greater reduction in
ments. Thus, MSC-derived therapeutics should be tested across the aging pro-inflammatory cytokine release (e.g. TNFα and IFNγ) when co-
spectrum, and experimental TBI studies should include cohorts of male cultured with BM-MSCs, than PBMCs from middle-age donors (56-60
and female aged animals (>15 months-old). years-old) in the same co-culture assays (Guan et al., 2019). Whether TBI
The assessment of MSC or secretome efficacy in TBI in aged animals in aged populations further impede these age-related impairments in
is poorly investigated with only few studies available. Tajiri et al. immunomodulatory capacity is unknown. Experimental studies in aged
(2014), showed efficacy of intravenous ASCs transplantation or their rhesus monkeys (16-26 years-old, which corresponds to 48-78 years in
secretome in young TBI rats (6 months-old), however, these treatments humans) subjected to cortical injury demonstrated that systemic
had reduced/no benefit in aged TBI rats (20 months-old). Specifically, administration of MSC-EVs improved motor function recovery after
secretome treatment improved neurological recovery and reduced brain injury (Moore et al., 2019). When compared to age-matched
anatomical damage in young TBI rats, but it was ineffective in aged TBI control subjects, aged monkeys treated with MSC-EVs had a signifi
rats. Incidentally, the treatment with the cellular counterpart ASC cant restoration of fine motor function of the hand, returning to pre-
induced improvement in sensorimotor and cognitive functions as well as operative grasp patterns and latency to retrieve a food reward in the
reduced anatomical damage in young and partially improved cognitive first three to five weeks of recovery (Moore et al., 2019). Follow-on
function recovery and anatomical damage in aged TBI rats, indicating an studies showed that MSC-EVs-treatment induces a phenotypic switch
age-dependent different responsiveness to MSC or secretome treatments of pro-inflammatory hypertrophic microglia toward an anti-
(Tajiri et al., 2014). When investigating ASC biodistribution, there was inflammatory ramified microglia state with homeostatic function (Go
reduced cell presence and survival in the spleen of aged TBI rats possibly et al., 2020). In addition, there was an increased ratio of ramified over
suggesting a reduced interaction with immune cells and lower immu hypertrophic microglia which correlated with faster motor function re
nomodulatory actions due to aging. Neurologic recovery observed after covery, thus supporting the role of MSC-EVs in reducing neuro
secretome systemic infusion can be driven by a direct effect of infused inflammation in aged rhesus monkeys following cortical injury.
9
Together, the preclinical studies suggest a partial resistance of the aged reported that hypoxia (5% O2) led to a significant increase in the
TBI brain to secretome treatment, and highlight the need for further expression of VEGF, BDNF, and NGF in dental pulp derived MSCs.
studies to identify immunomodulatory mechanisms in aged immune Overall, hypoxic cultures exhibited better stem cell morphology and had
cells in order to improve treatment options and boost secretome activity higher expression of stem cell markers (Ahmed et al., 2016).
for aged TBI patients. The use of Bioreactors could also represent an important tool to
further modulate the secretome. A small number of studies have used
1.5. Modulation of secretome profile bioreactors to alter MSC paracrine signaling for CNS regenerative
medicine purposes. Hupfeld et al. (2014) studied the effects of the
The characterization of the secretome profile is far from being fully expansion process of MSCs derived from the amniotic membrane (AM)
elucidated. Intrinsic non-defined features of MSC donors could and the umbilical cord (WJ-MSCs) on their biological characteristics.
contribute to the heterogeneity of the secretome profile (Teixeira et al., The culture of MSCs in controlled bioreactor systems led to less het
2016). For example, a proteomic analysis of the secretome of three erogeneity between cells from different donors. Moreover, cells
different aged-matched BM-MSC populations identified a different expanded in controlled suspension bioreactors significantly differed
secretome profile among each of them. Although data from in vitro ex from the cells cultured in flasks regarding surface markers, paracrine
periments and animal models indicate the degree of functional vari factors (e.g. VEGF, FGF-2, MCP-1, and SDF-1), and gene expression
ability is non-significant (Teixeira et al., 2015a, 2016), it may limit its profiles (Hupfeld et al., 2014). Interestingly, VEGF was only secreted by
application from a regulatory point of view. Additionally, the secretome WJ-MSCs, which had been expanded in bioreactors but not by cells
of MSCs isolated from different sources is itself different (Pires et al., expanded in flasks.
2016), leaving the question of which MSC source is best for each Computer-controlled stirred bioreactors have also been successfully
application. To answer these issues, different MSC application para used to modulate the secretome of hBM-MSCs (Teixeira et al., 2016). It
digms have been developed in recent years with the aim to control and was showed that cell expansion in this bioreactor system led to an
obtain more homogeneous MSC sources and secretome profiles. External enhancement of the neuroregulatory profile of the BM-MSC-derived
cues present in MSCs’ microenvironment also impact their paracrine secretome, particularly on the expression of cystatin C (Cys C), glia-
signaling. However, the mechanisms underlying the modulation of the derived nexin (GDN), Gal-1, pigment epithelium-derived factor
secretome by the external environment are still unknown. Harnessing (PEDF), and also BDNF, VEGF, NGF, and IGF-1 (Teixeira et al., 2016).
this knowledge would be extremely valuable to shape the secretome Additionally, it was possible to detect an upregulation of miRNA-16,
according to the context of the disease. The culture conditions used to typically found within BM-MSCs exosomes, and known to be involved
produce the secretome can be changed by regulating oxygen tension and in processes related to neurogenesis. The secretome produced under
including cytokines or growth factors (Kusuma et al., 2017). Addition dynamic conditions induced an increased differentiation of human
ally, the microenvironment can also be regulated by the implementation neural progenitor cells (hNPCs). When administered into the dorsal
of dynamic culture conditions such as porous scaffolds, scaffold-free ganglia of young adult rats, it was observed that the secretome of the
spheroids, or their combinations, as well as encapsulation in hydrogels bioreactor cultured BM-MSCs induced a higher differentiation of Ki67/
or even inside bioreactors (Sart et al., 2016). Examples of how it is DCX+ cells, indicating that it was more prone to induce neuronal dif
possible to modify the secretome are discussed below. ferentiation (Teixeira et al., 2016). All of these findings can be related to
MSC priming with inflammatory stimuli (e.g IFNγ and TNFα) is the the upregulation, in the secretome of dynamically cultured BM-MSCs, of
most commonly used method to stimulate the production of several anti- important regulators/immunomodulators of the neurogenic and neural
inflammatory factors in the secretome (Costa et al., 2021; Noronha et al., differentiation processes. Lam et al. (2021), testing a microcarrier-
2019). A detailed comparative proteomic analysis performed of five BM- spinner based bioreactor, showed differential expression pattern of
MSCs lines after 20 hours priming with INFγ identified 210 proteins with transcriptome, miRNAs and proteins in spinned compared to static MSC
significantly altered expression in secretome: 169 proteins were over cultures, enhancing pathways of extracellular matrix dynamics, cellular
expressed (e.g. IDO, PD-L1, ICAM-1, VCAM-1 and BST-2) while 41 were metabolism, differentiation potential, immunoregulatory function, and
downregulated (Guan et al., 2017). TNFα pre-conditioning of MSCs wound healing. The secretome is enriched for IL-6, IDO, TGF-β possibly
induced the upregulation of several immunomodulatory factors such as suggesting an immunomodulatory boost of MSCs under dynamic cul
PGE2, IDO and HGF (Prasanna et al., 2010), and in most cases a com tures (Lam et al., 2021; Mizukami et al., 2019). Allen et al. (2020)
bination of IFNγ and TNFα inflammatory cytokines for MSC priming is demonstrated that MSCs cultured in a continuous flow bioreactor pro
used to obtain synergistic effects (François et al., 2012; Szabó et al., duced more IL-6 and VEGF in the secretome overtime with a dose-
2015). dependent output. MSCs were responsive to an inflammatory cytokine
Oxygen tension conditions in the culture medium plays an important cocktail resulting in dynamic cytokine and growth factor secretion, with
role in MSC behavior. MSCs are usually cultured under normoxic con increased IL-6, VEGF and PGE2 release. Moreover, MSCs cocultured
ditions (21% O2). However, in the human body, these cells are exposed with activated PBMC, induced the inhibition of CD8+ T-cells and
to much lower concentrations of oxygen. Oxygen levels of 21% have decreased CD14highCD16low classical while increasing CD14highCD16high
been linked to DNA damage, leading to genomic instability and cellular monocyte intermediate populations. The monocytes secretion profile
senescence (Jagannathan et al., 2016). Several studies have reported was also altered with reduced TNFα and increased of IL-10 expression
that exposure to a hypoxic environment leads to changes in MSCs (Allen et al., 2020). Other groups confirmed the ability of bioreactor
physiology (Liu et al., 2013; Roemeling-van Rhijn et al., 2013; Yu et al., expanded MSCs to inhibit T cell proliferation via the upregulation of IDO
2013). The effects of a hypoxic environment also influence the secre and PD-L1 (Das et al., 2019; Lawson et al., 2017). To test the effect of
tome profile. In this regard, Teixeira et al. (2015b), showed that hypoxic vascular flow-induced shear stress on MSC signaling and function, Diaz
(5 % O2) and normoxic conditions induced alterations in the secretome et al. (2017) cultured MSCs in microfluidic systems, mimicking a uni
profile of Wharton Jelly (WJ) derived MSCs. The proteins expressed in form laminar flow. Under shear stress culture, MSCs increased the
both conditions were linked with neuroprotection, angiogenesis, and release of the antioxidant HO-1 and anti-inflammatory COX-2 media
neurite outgrowth processes, and their expression pattern varied ac tors, the latter associated with an increase production of PGE2. MSCs
cording to the oxygen concentration to which cells were exposed. cultured under shear stress exhibited enhanced potency in suppression
However, although differences were observed in the secretome of WJ- of TNF-α production by LPS-activated splenocytes, and reduced the
MSCs cultured under normoxic or hypoxic conditions, there were no amoeboid microglia activation in the hippocampus when infused
significant differences in their functional output (i.e. neuronal differ intravenously in rats 24h after TBI, (Diaz et al., 2017). These data sug
entiation) (Teixeira et al., 2015b). Similarly, Ahmed et al. (2016), gest that bioreactors may become a tool to empower secretome based
10
therapies. Baez-Jurado, E., Guio-Vega, G., Hidalgo-Lanussa, O., González, J., Echeverria, V.,
Ashraf, G.M., Sahebkar, A., Barreto, G.E., 2019a. Mitochondrial neuroglobin is
necessary for protection induced by conditioned medium from human adipose-
2. Conclusions derived mesenchymal stem cells in astrocytic cells subjected to scratch and
metabolic injury. Mol. Neurobiol. 56, 5167–5187. https://doi.org/10.1007/s12035-
Treatment of TBI patients has not changed much in the last 20 years, 018-1442-9.
Baez-Jurado, E., Hidalgo-Lanussa, O., Barrera-Bailón, B., Sahebkar, A., Ashraf, G.M.,
and no effective therapeutic options able to counteract injury progres Echeverria, V., Barreto, G.E., 2019b. Secretome of mesenchymal stem cells and its
sion are available. No single-agent treatment has been successfully potential protective effects on brain pathologies. Mol. Neurobiol. 56, 6902–6927.
translated to the clinical setting. Investigation into this has indicated the https://doi.org/10.1007/s12035-019-1570-x.
Beschorner, R., Nguyen, T.D., Gözalan, F., Pedal, I., Mattern, R., Schluesener, H.J.,
complicated and multifaceted nature of TBI as a critical cause for failure. Meyermann, R., Schwab, J.M., 2002. CD14 expression by activated parenchymal
It is conceivable that multiple therapeutic targets may need to be microglia/macrophages and infiltrating monocytes following human traumatic brain
addressed simultaneously to interfere with the natural evolution of brain injury. Acta Neuropathol. (Berl.) 103, 541–549. https://doi.org/10.1007/s00401-
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damage after TBI and improve patient’s outcome. MSC-derived secre Bustos, M.L., Huleihel, L., Meyer, E.M., Donnenberg, A.D., Donnenberg, V.S., Sciurba, J.
tome shows protection in experimental TBI, thus suggesting the poten D., Mroz, L., McVerry, B.J., Ellis, B.M., Kaminski, N., Rojas, M., 2013. Activation of
tial for a cell-free approach that would overcome important issues human mesenchymal stem cells impacts their therapeutic abilities in lung injury by
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related to intrinsic cell heterogeneity and safety concerns. https://doi.org/10.5966/sctm.2013-0033.
MSC-released factors have been identified as potent regulators of Carty, F., Mahon, B.P., English, K., 2017. The influence of macrophages on mesenchymal
immune cell phenotypes and functions. Every time a specific factor is stromal cell therapy: passive or aggressive agents? Clin. Exp. Immunol. 188, 1–11.
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inhibited, MSC efficacy is lost, pointing to a crucial role of that specific
Chang, C.-P., Chio, C.-C., Cheong, C.-U., Chao, C.-M., Cheng, B.-C., Lin, M.-T., 2013.
factor over the others. However, most of these observations have been Hypoxic preconditioning enhances the therapeutic potential of the secretome from
obtained using over simplified in vitro systems, in which only MSC- cultured human mesenchymal stem cells in experimental traumatic brain injury.
immune cell binary interactions are allowed. It is conceivable that in Clin. Sci. 124, 165–176. https://doi.org/10.1042/CS20120226.
Chen, Y., Li, J., Ma, B., Li, N., Wang, S., Sun, Z., Xue, C., Han, Q., Wei, J., Zhao, R.C.,
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synergistic effects of multiple mediators will be needed. MSCs and their microglia/macrophages in rat. Aging 12, 18274–18296. https://doi.org/10.18632/
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Chuang, T.-J., Lin, K.-C., Chio, C.-C., Wang, C.-C., Chang, C.-P., Kuo, J.-R., 2012. Effects
complex neurological disorders such as TBI. However, the challenge of secretome obtained from normoxia-preconditioned human mesenchymal stem
going forward is to find the best cocktail of MSC-released bioactive cells in traumatic brain injury rats. J. Trauma Acute Care Surg. 73, 1161–1167.
factors that will confer robust long-term neuroprotection and improve https://doi.org/10.1097/TA.0b013e318265d128.
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Schneider, J., Vizoso, F.J., 2021. Functional heterogeneity of mesenchymal stem
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Financial support was provided from the Ministero Della Salute Cell. Mol. Life Sci. CMLS 78, 447–467. https://doi.org/10.1007/s00018-020-03600-
(Italy) within the framework Ricerca Finalizzata GR-2016-02361904, 0.
Cox, A.L., Coles, A.J., Nortje, J., Bradley, P.G., Chatfield, D.A., Thompson, S.J.,
from the PORTUGAL 2020 Partnership Agreement, from the European
Menon, D.K., 2006. An investigation of auto-reactivity after head injury.
Regional Development Fund (ERDF) FEDER (Portugal), and from the J. Neuroimmunol. 174, 180–186. https://doi.org/10.1016/j.jneuroim.2006.01.007.
Foundation for Science and Technology (FCT) - project UIDB/50026/ Cui, L., Saeed, Y., Li, H., Yang, J., 2022. Regenerative medicine and traumatic brain
2020 and UIDP/50026/2020 (Portugal) and scholarship PD/BDE/ injury: from stem cell to cell-free therapeutic strategies. Regen. Med. 17, 37–53.
https://doi.org/10.2217/rme-2021-0069.
143149/2019’. Daglas, M., Draxler, D.F., Ho, H., McCutcheon, F., Galle, A., Au, A.E., Larsson, P.,
Gregory, J., Alderuccio, F., Sashindranath, M., Medcalf, R.L., 2019. Activated CD8+
Declaration of Competing Interest T cells cause long-term neurological impairment after traumatic brain injury in mice.
Cell Rep. 29, 1178–1191.e6. https://doi.org/10.1016/j.celrep.2019.09.046.
Dai, X., Zhu, B.T., 2009. Suppression of T-cell response and prolongation of allograft
The authors declare that they have no known competing financial survival in a rat model by tryptophan catabolites. Eur. J. Pharmacol. 606, 225–232.
interests or personal relationships that could have appeared to influence https://doi.org/10.1016/j.ejphar.2008.12.053.
Dang, J., Yang, J., Yu, Z., Chen, L., Zhang, Z., Wang, K., Tang, J., Yi, C., 2022. Bone
the work reported in this paper. marrow mesenchymal stem cells enhance angiogenesis and promote fat retention in
fat grafting via polarized macrophages. Stem Cell Res. Ther. 13, 52. https://doi.org/
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Experimental Neurology 357 (2022) 114199

Contents lists available at ScienceDirect

Mesenchymal stromal cell secretome for traumatic brain injury: Focus on

When Ngb is silenced secretome efficacy is reduced

When Ngb is silenced secretome efficacy is reduced

Chuang Human 24h // 25x Adult male • 500 μg At 3 days: At 3 days: //

Fig. 1. MSC secreted factors that alter im

Fig. 2. MSC secreted factors with immune

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1 s2.0 S0014488622002242 Main

Uploaded by

Copyright:

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1 s2.0 S0014488622002242 Main

Uploaded by

Document Information

Original Title

Copyright

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Share this document

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1 s2.0 S0014488622002242 Main

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Copyright:

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Experimental Neurology 357 (2022) 114199

Contents lists available at ScienceDirect

Mesenchymal stromal cell secretome for traumatic brain injury: Focus on

When Ngb is silenced secretome efficacy is reduced

When Ngb is silenced secretome efficacy is reduced

Chuang Human 24h // 25x Adult male • 500 μg At 3 days: At 3 days: //

Fig. 1. MSC secreted factors that alter im­

Fig. 2. MSC secreted factors with immune

You might also like

Fig. 1. MSC secreted factors that alter im