Mazziotti et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03193-6(2024) 21:209

Journal of Neuroinflammation

Open Access

REVIEW

The contribution of tumor necrosis
factor to multiple sclerosis: a possible rolein progression independent of relapse?
Valentina Mazziotti1, Francesco Crescenzo2, Ermanna Turano1, Maddalena Guandalini1, Maddalena Bertolazzo1,
Stefano Ziccardi1, Federica Virla1, Valentina Camera1, Damiano Marastoni1, Agnese Tamanti1 andMassimiliano Calabrese1*

Abstract
Tumor necrosis factor (TNF) is a pleiotropic cytokine regulating many physiological and pathological immunemediated processes. Specifically, it has been recognized as an essential pro-inflammatory cytokine implicatedin multiple sclerosis (MS) pathogenesis and progression. MS is a chronic immune-mediated disease of the central
nervous system, characterized by multifocal acute and chronic inflammatory demyelination in white and grey matter, along with neuroaxonal loss. A recent concept in the field of MS research is disability resulting from Progression
Independent of Relapse Activity (PIRA). PIRA recognizes that disability accumulation since the early phase of the diseasecan occur independently of relapse activity overcoming the traditional dualistic view of MS as either a relapsinginflammatory or a progressive-neurodegenerative disease. Several studies have demonstrated an upregulation in TNF
expression in both acute and chronic active MS brain lesions. Additionally, elevated TNF levels have been observedin the serum and cerebrospinal fluid of MS patients. TNF appears to play a significant role in maintaining chronic
intrathecal inflammation, promoting axonal damage neurodegeneration, and consequently contributing to diseaseprogression and disability accumulation. In summary, this review highlights the current understanding of TNF and its
receptors in MS progression, specifically focusing on the relatively unexplored PIRA condition. Further research in thisarea holds promise for potential therapeutic interventions targeting TNF to mitigate disability in MS patients.
Keywords TNF biology, MS pathology, PIRA, MS lesions, Chronic compartmentalized inflammation,
Neurodegeneration, Biomarkers, Disease progression*Correspondence:
Massimiliano Calabresemassimiliano.calabrese@univr.it
1
Neurology B Unit ‑ Multiple Sclerosis Center, Departmentof Neurosciences, Biomedicine and Movement Sciences, Universityof Verona, 37134 Verona, Italy
2
Neurology Unit ‑ Multiple Sclerosis Center, Scaligera Local Unit of Healthand Social Services 9, Mater Salutis Hospital, 37045 Legnago, Verona, Italy

Introduction
Multiple sclerosis (MS) is a chronic immune-mediatedand neurodegenerative disease of the central nervous
system (CNS) affecting millions of people worldwide [1]and it is the most common cause of non-traumatic neurological disability in young adults [2].
MS is a complex multifactorial disease caused by complex gene–environment interactions and characterized by multiple pathological hallmarks, ranging from
immune dysregulation and neuroinflammation to neurodegenerative mechanisms [3].

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Mazziotti et al. Journal of Neuroinflammation(2024) 21:209

Several molecular changes, including increases incytokines, chemokines, nitric oxide, reactive oxygen species, glutamate, and free radicals, affect the pathogenesis
and the course of MS [4].
The clinical course of MS is highly variable, heterogeneous, and unpredictable at the individual level. Generally,
it is characterized by transient and recurrent episodes of
focal acute CNS inflammation early on, with complete orpartial resolution (relapsing–remitting MS–RRMS) and,over time, by a prominent process of neurodegeneration,
resulting in a late, slow, steady, progressive accumulationof physical disability and cognitive impairment (secondary progressive MS–SPMS) [5]. On the other hand, a
gradual and continuous neurological decline from thedisease’s onset characterizes the MS subtype known asprimary progressive MS (PPMS) [5–7].
Beyond this traditional phenotypic categorization, it is
now clear that MS progresses along a continuum fromRRMS to progressive MS (PrMS), with distinct levels ofneurologic reserve explaining phenotypic differences [8].
This emerging view of MS as a single-stage disorder inwhich all patients exhibit a progressive course since disease onset, which can overlap with relapses [6, 8], is supported by the new concept of progression independent ofrelapse activity (PIRA) [9]. The term PIRA, proposed by
Kappos et al., refers to the progressive clinical deterioration occurring in many RRMS patients without signs ofinflammatory activity [9]. This notion aligns with several
previous observational studies showing that disability
accumulation is largely independent of superimposed
focal inflammation, referring to additional inflammatory
lesions, and is undetectable by conventional clinical-radiological parameters [10–13].
Although the frequency of PIRA has been reported
within the first 5 years following the first MS-related clinical attack, its identification in clinical practice remains
unclear due to the lack of standardized definitions (suchas a time window after the last relapse) and/or measures
to detect it (such as based on the expanded disability
status scale—EDSS—score or an increase in composite
measures) [14].
The mechanisms driving PIRA have yet to be fully elucidated but are undoubtedly associated with smouldering
inflammatory and neurodegenerative processes. In a prospective, large sample size study, Cagol et al. showed that
RRMS patients with PIRA (defined as a 6-month confirmed disability progression with no relapse during the
90 days before and the 180 days after the initial increasein the EDSS score) exhibit more pronounced diffuse cerebral cortical volume loss [15]. This finding aligns withseveral studies demonstrating that grey matter (GM)atrophy is predictive of long-term physical and cognitive
disability [16] and conversion to PrMS [17].

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Cerebral GM damage, which manifests as both focal
cortical lesion(s) and diffuse cortical and deep GM atrophy, provides one of the best clinical correlations withirreversible disability accumulation [16, 18] and it is
topographically associated with aberrant tertiary B-cellenriched lymphoid structures affecting the cerebral
meninges [19]. The extent of meningeal immune infiltration is correlated with the degree of subpial GM demyelination, microglial activation, and axonal loss [19–22].
MS patients with a progressive and severe course of
the disease also display chronic active lesions (CALs), a
subset of white matter (WM) lesions characterized by aninactive core surrounded by a “rim” of activated microglia [23–25]. CALs are associated with nearby persistentdemyelination and axonal loss, even in the absence ofblood‒brain barrier (BBB) damage [23–25].
Molecular-neuropathological studies on progressive
MS patients supported the hypothesis that soluble factors (chemokines and cytokines) produced by meningeal tertiary lymphoid structures and/or circulating
immune cells may diffuse throughout the cerebrospinal
fluid (CSF) into the cortex, inducing brain damage either
directly or indirectly through microglial activation [26].
In this regard, Kosa and colleagues found that CSF biomarkers associated with immune-related pathways correlate with clinical and imaging MS severity outcomes andpredict future disability [27].
All these findings suggest that chronic inflammation in
the CNS continuously disturbs neuroaxonal homeostasis, leading to prominent neurodegeneration, even outside of MS relapses, especially at the progressive stage[28]. This confirmed that compartmentalized inflammation (involving the CSF, meninges, and parenchyma) is amajor mechanism driving progressive multiple sclerosis.
Among the different cytokines found to increase in the
CSF of MS patients [26], tumor necrosis factor (TNF)
represents one of the main proinflammatory cytokines
correlated with the degree of disability in patients with
progressive MS [29].
Selmaj et al. also provided significant evidence that anincrease in TNF occurred locally within the CNS of MS
patients, specifically in acute and chronic active MS brain
lesions [30]. This further suggests that the combination of
inflammation, demyelination and neurodegeneration is ahighly specific process in MS, as supported in a study by
Fischer et al. [31].
TNF exerts its potent proinflammatory activity by activating two specific TNF receptors (TNFRs): TNF receptor type-1 (TNFR1) and type-2 (TNFR2) signaling [32,33]. In addition to this inflammatory action, TNF has
excitotoxic [34] and necro-apoptotic effects on oligodendrocytes [35, 36] and neurons mainly through TNFR1
activation [37].

Mazziotti et al. Journal of Neuroinflammation(2024) 21:209

A post-mortem study revealed that an imbalance of
TNF receptor type-1 (TNFR1) and type-2 (TNFR2)
signaling plays a role in determining the severity of MS[38], demonstrating a strong correlation between compartmentalized inflammation and the high expression ofgenes involved in the TNFR1 signaling cascade [38].
This comprehensive review explores the potential
impact of TNF pathway alterations on MS progression
and the potential of selective targeting and detection of
TNF-TNFRs, specifically focused on the PIRA condition,
on which many questions persist regarding its frequency,
pathological determinants, treatment, and implications[39]. Therefore, understanding the role of TNF signaling in PIRA could shed light on the neurodegenerative
mechanisms that drive the progression of RRMS fromthe earliest stages of the disease [39].

TNF biology, cellular production and signaling
pathways
The master proinflammatory cytokine TNF has beenshown to have a broad spectrum of cellular effects,
including inflammatory response, cellular activation, andprogrammed cell death [40]. TNF belongs to the TNF
superfamily, which includes 19 ligands produced primarily by monocytes/macrophages but also by T and B lymphocytes, smooth muscle cells, adipocytes, osteoclasts,
and fibroblasts, although in smaller quantities [40, 41].
TNF is expressed initially as a transmembrane protein(mTNF, 26 kDa 233-amino-acid), which requires proteolytic cleavage by the TNF converting enzyme (TACE)
to release soluble TNF (sTNF, 17 kDa 157-amino-acid).
mTNF and sTNF are produced by a wide range of
peripheral and central immune cells, such as activatedmacrophages, effector CD4 + and CD8 + T cells, B lymphocytes and microglia, as well as neurons, oligodendrocytes, and astrocytes [42].
Both mTNF and sTNF are biologically active and exert
their effects by modulating a complex signaling pathway
with wide-ranging downstream responses through two
distinct surface receptors belonging to the TNFRs-superfamily (comprising 29 receptors): the TNF receptorsuperfamily member 1A (TNFRSF1A-TNFR1; p55/60;
CD120a) and TNF receptor-superfamily member 1B(TNFRSF1B-TNFR2; p75/80; CD120b) [40–42].
The two receptors differ significantly in structure, binding affinity, localization, function and activation of signaling pathways [43, 44].
TNFR1, which is expressed on the membrane of allcell types except for erythrocytes, shows a high affinity
for sTNF, promoting both necrosis and apoptotic pathways as well as proinflammatory signaling [45] throughits death domain (DD), which, when activated by TNF
binding, recruits the TNFR1-associated death domain

Page 3 of 14(TRADD). TRADD can in turn recruit Fas-associateddeath domain (FADD) and receptor-interacting serine/
threonine-protein kinase 1 (RIPK1), which can either
lead to necroptosis through RIPK3 and mixed lineage
kinase domain-like pseudokinase (MLKL) activation or
apoptosis through caspase 8 and caspase 3 recruitment[46, 47]. In contrast, proinflammatory signaling is mediated by TNFR-associated factor 2 (TRAF2) activationof mitogen-activated protein kinases (MAPKs), such as
c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the transcription factor nuclear
factor-κB (NFκB) [46].
TNFR2, which is expressed only in a few cell types(neurons, oligodendrocytes, microglia, and T lymphocytes), mediates local homeostatic effects, such as cell
survival, tissue regeneration and inflammation, by preferentially binding to mTNF [48, 49]. Unlike TNFR1,
TNFR2 does not have a death domain. Nevertheless, arecent study has shown that under some circumstances,
TNFR2 signaling also has pro-apoptotic effects by amplifying TNFR1-mediated stimulation of apoptosis orcooperating in the binding of TNF to TNFR1 [42, 50].
However, the mechanism of TNFR2-mediated cell deathis still unclear, and homeostasis and cell survival remain
the primary functions exerted by TNFR2-mediated signaling through TRAF (1/2) activation of MAPKs (JNK and
ERK), protein kinase B (Akt), and NFκB [46].
Therefore, albeit in different ways, both TNFR1 andTNFR2 signaling may lead to NF-κB and MAPK activation, increasing the expression of inflammatory genes
encoding chemokines and cytokines (including TNF
itself ) [43, 44] and inducing antiapoptotic transcriptional
programs that promote cell survival, cell proliferationand cell differentiation [51, 52].
This duality of TNFR signaling, which can induce cell
survival and cell death, depends on the cellular environment, the relative surface levels of TNFR1 and TNFR2,
and their cellular activation status (Fig. 1). However, theeffects of altering the TNFR1/TNFR2 balance under normal and altered physiological conditions remain unclear[53].

Potential pathological implications of TNF‑TNFRs
impairment in MS and EAE
Several studies on human and experimental MS havedemonstrated the involvement of TNF in various pathological hallmarks of MS, including neuroinflammation,neurodegeneration and demyelination.
The role of TNF in neuroinflammation associated with MS

TNF plays a crucial role in several immune-mediatedconditions, including rheumatoid arthritis [54], systemic lupus erythematosus [55] and Crohn’s diseaseMazziotti et al. Journal of Neuroinflammation(2024) 21:209

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Fig. 1 TNF signaling. TNF signaling is mediated by two isoforms (mTNF and sTNF), which exert their effects by modulating a complex signaling
pathway through two distinct surface TNF receptors: TNFR1 and TNFR2. TNFR1 shows a high affinity for sTNF, which once bound, recruits TRADD.
TRADD binds to FADD and RIPK1, leading to necroptosis through RIPK3 and MLKL activation, or apoptosis through caspase 8 and caspase 3
activation. On the other hand, mTNF interacts with TNFR2, inducing inflammation and homeostasis through TRAF1/2 activation of JNK, ERK MAPKs,
Akt and/ or NFκB[56]. As a potent mediator of inflammation, principally via TNFR1 signaling, TNF is considered one of
the major cytokines involved in the pathogenesis of MS[32, 33].
A relevant action of TNF is to activate T lymphocytes, enhancing their proliferation and recruitment
and increasing proinflammatory cytokine productionin the CNS by inducing the activation of NF-κB signaling pathways [57]. TNF-dependent T-cell activation
contributes to blood‒brain barrier (BBB) damage via
secondary meningeal mast cell activation and therefore
promotes further inflammatory cell influx with consequent myelin and neuronal damage [58, 59].
Not surprisingly, elevated TNF production is foundin MS patients [30, 60] and in experimental autoimmune encephalomyelitis (EAE), the most commonlyused murine model of MS [60].
High TNF levels are found in active demyelinating
lesions [29] and in the serum and CSF of MS patients[61–63], in correlation with the increase in the degreeof disease severity [63–66]. In EAE mice, TNF mRNA
expression is upregulated in the CNS in parallel withdisease progression, and its exogenous administration
increases EAE severity [60, 67] since it is involved in
immune cell (macrophage and T cell) activation andinfiltration into the CNS [66].

The use of EAE transgenic mice for TNF and TNFRs
has significantly contributed to understanding the pathological role of TNF in MS [49].
Compared with wild-type (WT) EAE mice, TNF-gene
knockout (KO) EAE mice exhibit a milder disease course
due to reduced leukocyte intrathecal trafficking and BBB
permeability [68]. This evidence suggests that TNF signaling alterations are involved in the (early) pathological
MS mechanisms that occur in the CNS [68].
In addition to cytokines, several studies have investigated the role of TNFRs in MS pathology. Specifically,
compared with WT EAE mice, TNFR1 KO EAE mice
showed a reduction in immunopathological signs andsymptoms of the disease, whereas TNFR2 KO EAE mice
showed more severe disease symptoms, enhanced T-cell
infiltration in the CNS and diffuse demyelination [69].
Intriguingly, TNFR2 has recently been demonstrated
to be crucial in regulating T-cell biology [69]. Specifically,
it is known to be expressed by regulatory T cells (Tregs)
and is involved in their proliferation and expansion [70].
­Tregs are a specialized subpopulation of T cells that act
to suppress immune response, inhibiting T cell proliferation and cytokine production [71], ­Tregs are essential
for maintaining immune homeostasis and preventing
autoimmunity [67–74]. Not surprisingly, impaired functional suppression of T
­ regs in response to autoreactive T

Mazziotti et al. Journal of Neuroinflammation(2024) 21:209

cells is typically reported in MS [75]. In line with this, arecent study on T
­ reg-restricted TNFR2-deficient micewith induced EAE revealed that these mice developed
an aggressive disease, indicating the critical protectiverole of TNFR2 signaling [76]. However, the significanceof intrinsic TNFR2 signaling in T
­ reg cells in vivo remains
incompletely defined [76].
These findings support the critical role of TNFR1 signaling in the induction of a proinflammatory environment in the CNS [68]. In contrast, TNFR2 appears to be
involved in neuroprotection and repair processes [68].
The role of TNF in neurodegeneration, demyelination
and remyelination associated with MS

In addition to immune cell activation and infiltration,
TNF signaling engages in neurodegenerative processes.
TNF promotes neuronal excitotoxicity and oligodendrocyte death, acting directly on neurons and glial cells
through TNFRs, with further TNF release [46, 77].
An elegant study by Centonze et al. showed that
increased concentrations of TNF released by activatedmicroglia induce changes in the expression and physiological properties of glutamate AMPA (α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) receptors(AMPRs) and NMDA (N-methyl-D-aspartate) receptors(NMDARs) in EAE mice [78]. Specifically, TNF acting
on neuronal TNFR1 receptors causes excitotoxicity byincreasing the surface expression of AMPARs and theactivation of NMDARs, prolonging the duration of theglutamate postsynaptic response [78].
In addition to excitotoxicity, TNF-TNFR1 signaling is
involved in triggering oligodendrocyte apoptosis [35].
Consistent with this, TNF-overexpressing transgenic
mice developed spontaneous demyelinating lesions likethose observed in MS [35, 79, 80]. On the contrary, TNF
through TNFR2, facilitates remyelination by promoting
oligodendrocyte differentiation in EAE [81]. Furthermore, TNFR2-KO mice develop more severe EAE motor
disease than WT mice [81–83]. In TNFR2 conditional
KO EAE mice, a novel transgenic mouse with selective
TNFR2 ablation in oligodendrocytes, altered TNFR2
signaling results in impaired remyelination [83].
These findings suggest that TNF could exhibit abimodal effect, depending on the receptor it binds to,thereby triggering distinct signaling pathways. In MS, theTNFR1 signaling cascade plays a harmful role, whereas
TNF acting via TNFR2 exerts a protective effect, attenuating the disease’s aggressive course. Enhancing TNFR1
and weakening TNFR2 signaling, the TNF contributes to
MS pathogenesis and progression, leading to inflammatory demyelination, remyelination failure, and neuronal
functional damage, including synaptic impairment.

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Increased intrathecal TNF expression and impaired
TNF‑TNFRs signaling in association with meningeal
inflammation in MS
Increased levels of proinflammatory cytokines and cytotoxic mediators are found in the CSF of MS patients [26];specifically, CSF levels of TNF are correlated with thedegree of disability in patients with PrMS [26, 29, 84,85] but are not detected in patients with other neurodegenerative diseases [30]. This increase is determined byimmune cell infiltration into the CNS; in particular, lymphocytes and macrophages enter the brain through theperivascular space and meninges, where they can releasecytokines and chemokines that trigger glial cells andneurons to produce additional inflammatory mediators,
such as IL-1β, TNF, and IFN-ɣ [86]. Mounting intrathecal neuroinflammation induces a local and chronic
immune response that alters synaptic transmission andneuroaxonal homeostasis [20], leading to an increasingly
inflammatory environment in the CSF, which bathes thecortical layers [26, 84]. In this regard, a strong correlation
was found between CSF/meningeal inflammation andthe degree of cortical damage, microglial activation, andaxonal loss [20, 26, 84]. Chronic inflammation causes GM
damage in MS from the earliest stages of the disease. This
leads to disability accumulation independent of acuteinflammation due to the decreased capacity of the compensatory mechanisms, including neuroplasticity, remyelination, redistribution of sodium channels along axons
to maintain nerve conduction and expression of neurotrophic factors to support neuron survival and repair, as
well as immune modulation [20, 28, 87–89]. Early cortical GM damage is indeed related to a more severe andrapid disease course in terms of disability progression
and cognitive impairment [28].
In this regard, it has been demonstrated that neuroinflammation increases with MS progression, identifying specific inflammatory pathways correlated with MS
progression, which include both the innate and adaptive
immune pathways of T helper (Th) 17 (IL17, GM-CSF
and IL6), Th1 (IFNγ and TNF) and Th2 (IL13 and IL4)
phenotypes [27]. Moreover, Magliozzi et al. showed thatmeningeal inflammation specifically alters the balance
between TNFR1 pro-cell death and TNFR2 pro-cell survival signaling, causing more severe disease manifestations from the early stages [38]. In addition, this study
not only confirmed elevated TNF levels in the CSF of MS
patients at the time of diagnosis but also revealed greater
TNFR1 gene overexpression in MS patients, especiallyin the cortical GM tissues of progressive disease patients[38]. These results are in line with a recent study by Piconand colleagues that provides substantial evidence for
TNF-mediated activation of necroptotic signaling via
TNFR1 in cortical neurons of progressive MS patients

Mazziotti et al. Journal of Neuroinflammation(2024) 21:209[37]. In fact, the study demonstrated increased expression of multiple steps in the TNF-TNFR1 signaling pathway leading to necroptosis, including the key proteins
TNFR1, FADD, RIPK1, RIPK3 and MLKL [36].
All these results support the hypothesis that neurodegeneration in MS is mainly driven by chronic inflammation in the CNS, with a preponderant involvement ofactivated TNF–TNFR1 signaling.

Evidence for TNF involvement in MS lesion
formation
Degenerative processes include demyelination, axonal
injury, and neuron loss, and result in multifocal WM
lesions and diffuse GM damage in subpial and subventricular regions close to the CSF and meninges [90]. Significant upregulation of TNF and TNFR1 was found inwhite matter (WM) and subpial GM lesions [81].
WM lesions can be classified as active, chronic active(CALs; smouldering, slowly expanding, mixed active/
inactive), remyelinating, or chronic inactive lesions [92].
Active lesions develop from normal-appearing whitematter (NAWM) and are characterized by areas of demyelination and activated macrophages and microglia.
These lesions can remyelinate in the presence of activated microglia or evolve into CALs or inactive lesions[93]. CALs exhibit a demyelinated hypocellular nucleus
and rims of iron-laden activated microglia [94, 95], while
inactive lesions are well-defined areas of demyelination
and axonal degeneration in the absence of inflammation[23, 80, 93].
Chronic compartmentalized inflammation leads to theformation of CALs, which increase in number as the disease progresses [31, 96, 97]. In fact, they represent morethan half of all focal WM lesions, especially in progressive MS patients [98], depicting a relevant pathologicalfinding associated with a severe disease course mediatedby neuroaxonal damage in the absence of superimposed
acute inflammatory activity [23, 94, 97].
The presence of TNF in CALs and its absence in inactive lesions is a noteworthy finding consistent with previous observations indicating immunoreactivity primarilyin activated microglia and T cells at the lesion edge [30,95]. In addition, a seminal study by Jackle et al. exploredthe immunological-molecular profile of CALs; throughmicroarray analysis, they found an upregulation of different genes associated with immune functions, including
those for TNF and its receptors, indicating its significant
role in the formation of CALs [99]. Specifically, the transcript expression of the TNFR1 gene increased almost
fivefold in these lesion types [99]. These results suggest
an ongoing inflammatory response associated with the
TNF and TNFR1 overexpression in CALs, which contributes to the exacerbation of MS outcomes.

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Furthermore, TNF and TNFR1 levels also increasein cortical lesions [38]. GM damage, including cortical lesions and atrophy, is already present in the earlyphase of MS [28, 80, 100, 101] and becomes more prominent during the disease progression [102]. Early corticalinvolvement is related to a more severe and rapid disease course in terms of disability progression and cognitive impairment. The transcriptional profile of chronic
subpial GM lesions isolated from MS brain samples
with prominent meningeal inflammation, revealed an
upregulation of TNFR1 and genes encoding caspase 1,
proinflammatory cytokines and chemokines, consistently with skewing toward a detrimental environment
and proinflammatory microglia phenotype within theselesions [91]. This evidence is also supported by the studyof Magliozzi et al., which demonstrated that in subpial
GM lesions of progressive MS patients, TNFR1, and not
TNFR2, was exclusively increased [38]. Overall, thesestudies highlight TNF-TNFR1 signaling as a potential
future therapeutic target for mitigating the impact ofboth CALs and GM lesions in MS.

Exploring the potential role of TNF signaling
in the emergence of PIRA
According to the newly proposed categorization, the different clinical MS phenotypes (RRMS, SPMS, PPMS,
and PRMS) identified in 1996 [103] are summarized inrelapsing–remitting disease versus progressive disease[104]. Both clinical forms of MS appear to reflect thesame underlying disease process characterized by neuroinflammation and subsequent neurodegeneration [105,106] present in all MS lesions across the entire disease
course [105–110]. In this context, compartmentalizedneuroinflammation appears crucial for the onset andprogression of neurodegenerative mechanisms that resultin axonal loss and brain atrophy [111], which are strongly
correlated with long-term functional and cognitive disability [112].
Several studies have also proven the association
between focal inflammatory activity and diffuse andregional atrophic changes [15, 113, 114]. Specifically, MS
lesions cause brain volume loss through direct inflammatory damage leading to myelin and axonal loss and, indirectly, tissue loss following Wallerian degeneration [15].
In addition, evidence from neuropathological, imaging,and biomarkers studies suggests more continuous axonal
loss across all clinically defined stages of MS, both inearly and relapsing MS rather than in more advanced andprogressive stages [9].
The classic RRMS/PrMS subdivision has been overcome since the emergence of a new concept of MS, evidence of progression independent of relapse activity(PIRA) [15].

Mazziotti et al. Journal of Neuroinflammation(2024) 21:209

PIRA represents the first and main event responsiblefor irreversible disability accumulation in adult patientswith RRMS, which occurs in 80% to 90% of patients [9].
PIRA is already present in the early phases of disease andmay even occur during disease-modifying treatments(DMTs) [15, 111, 112, 115]. Two similar important studies investigating PIRA in early MS patients showed thatapproximately one-fourth of patients with RRMS may
develop PIRA during the first ten years of the disease [14,116]. Patients who developed their first PIRA event very
early in the disease course had an unfavourable prognosis[14].
PIRA occurs in approximately 5% of all patients with
RRMS annually, causing at least 50% of all disability
accrual events in typical RRMS [117]. In this regard, a
recent study confirmed that up to 50% of disability accumulation in adult patients with RRMS is not associatedwith evident relapses [118]. Relapses may mask diseaseprogression, and the gradual loss of function might gounnoticed by some patients and their physicians; thiswould explain why PIRA is underestimated in patientswith RRMS [118].
Furthermore, patients with PIRA show significantly
increased GM atrophy and a greater number of CALs,
providing additional important evidence of the association between PIRA and diffuse neurodegeneration [15].
In PIRA, neuroinflammation is associated with several
pathological processes, such as brain atrophy, failure ofcompensatory mechanisms and impaired remyelination[119, 120]. Understanding molecular mechanisms underlying TNF signaling and associated neuroinflammation in
PIRA is crucial for developing targeted therapies to slow
the MS progression and potentially identifying prognostic and predictive disease values associated with TNFTNFRs levels.
Currently, there are no specific biomarkers for identifying PIRA conditions. Overall, the only biomarker ofongoing neuronal damage considered is serum neurofilament light chain (sNfLs). However, its association withlong-term clinical outcomes or its ability to reflect slow
and diffuse neurodegenerative damage in MS is not completely clear [121]. This lack of clarity is probably due tounstable measurements subject to physiological changes
such as age or body mass index fluctuations [121, 122].
Although early treatment with DMTs delays the diagnosis of progression over time [123], the ability to target
PIRA remains an unmet need, even during highly effective treatments [9, 117]. Nevertheless, several recent
observational studies failed to confirm a beneficial association of DMT with PIRA [117, 124, 125].
Hence, identifying a biological target that specificallyreflects current and future prognostic disability andPage 7 of 14

irreversible CNS tissue damage due to PIRA is urgently
needed.

Anti‑TNF therapy and its potential use for PIRA
Based on the strong proinflammatory activity of the TNF,
several anti-inflammatory drugs targeting TNF signaling
have been developed and approved for treating inflammatory diseases, such as Crohn’s disease, ankylosing
spondylitis, and rheumatoid arthritis. Specifically, five
TNF blockers are available for clinical use: infliximab,
adalimumab, golimumab, certolizumab and etanercept.
Anti-TNF serum is composed of either anti-TNF antibodies (infliximab, adalimumab, golimumab and certolizumab) or TNFR fusion proteins (etanercept) that act
as antagonists by blocking TNF (both mTNF and sTNF)
interactions with TNFRs [126]. Despite being consideredrelatively safe and effective for the above-mentioned diseases, severe effects associated with immune suppression
have been reported in MS [66, 127]. In particular, a clinical trial of infliximab showed unfavourable results, withincreased disease activity and MRI lesion load, proving
the association of TNF inhibitors with CNS demyelination [127, 128]. Although the relationship between TNF
blockers and demyelination remains uncertain, it is likelythat these blockers are not selective, i.e., they block theinteraction between TNFR1, which has a primarily proinflammatory effect, and TNFR2, which has a primarily
protective effect. This finding confirms the crucial andcontroversial role of TNF in the CNS, which exerts bothpotent proinflammatory effects (via TNFR1) and essential protective functions (via TNFR2) under pathologicalconditions [31, 66]. Specifically, TNF through TNFR2
signaling modulates the reactivity of self-reactive T cells
to self-antigens, promoting the expansion of Treg cells
and, subsequently, the preservation of myelin oligodendrocytes [90]. Selective inhibition of TNFR1 and selective
activation of TNFR2 through the use and even discoveryof new antagonist and agonist antibodies could represent a new molecular target for developing therapeutic
agents for MS [49]. A recent preclinical study showedthat atrosab, a human monovalent antibody selectively
against TNFR1 developed for treating inflammatory diseases, reduces disease severity. This preclinical evidence
seems promising for finding novel effective drugs for MS
and perhaps PIRA in the future [32].
The impact of DMTs on TNF‑TNFRs serum levels
in patients with PrMS
Peripheral TNF levels are elevated in PPMS patientsand correlate with disease progression, while results for
RRMS patients are inconclusive [49, 129]. The detection of soluble TNFRs (sTNFR1 and sTNFR2) in serumMazziotti et al. Journal of Neuroinflammation(2024) 21:209

has been suggested as a potential prognostic marker for
PrMS [129, 130].
Consequently, researchers have focused on serumdetectable TNF-signaling to attempt to distinguish MS
forms, monitor disease activity and assess treatment
responses [49, 129, 130].
Several studies have shown that commonly used MS
drugs can indirectly modulate TNF and sTNFRs expression [131–137]. Novel and increasingly effective DMTs
approved in the last decades significantly impact theimmune system, including TNF expression [131, 132].
These DMTs, including oral fumarates, glatiramer acetate (GA), teriflunomide and selective sphingosine 1‐
phosphate receptor (S1PR) modulators (Fingolimod
and Siponimod), as well as cell‐depleting therapies suchas cladribine, anti‐CD20 (Ocrelizumab) and anti‐CD52
monoclonals (Alemtuzumab), reduce disease activity anddisability progression, albeit in a different way [132]. GA
treatment promotes Th2 and Treg expansion, releasing
neurotrophic factors and anti-inflammatory cytokines,
and, conversely, reducing pro-inflammatory mediators
like TNF and TNFRs [133]. Teriflunomide, a chemotherapeutic agent, inhibits the proliferation of B cells, T cells,
and macrophages by inserting itself into DNA strands,
and selectively suppresses pro-inflammatory cytokines
expression, such as TNF [134]. S1PR modulators sequester autoreactive lymphocytes within lymph nodes, preventing their infiltration into the CNS [135]. They also
inhibit T cell differentiation into pro-inflammatory
Th1 and Th17 cells, producing TNF [135]. Specifically,
Fingolimod-treated mature dendritic cells (DCs) show
impaired phagocytic capacity and down-regulated several pro-inflammatory cytokines, including TNF [136].
Lastly, a recent study by Nowak-Kiczmer et al. found that
serum levels of sTNF-R1 and sTNF-R2 significantly differed between PPMS patients treated with Ocrelizumab
and treatment-naïve progressive patients, with highersTNF-R2 levels in the treated group [137].
By modulating TNF expression, DMTs help reduce
inflammation and potentially slow down disease progression. This modulation can have significant consequences
for PIRA, as it may help mitigate some of the pathological processes associated with the condition, such as
brain atrophy, failure of compensatory mechanisms, andimpaired remyelination.

Discussion
TNF plays a pivotal role in the pathogenesis of MS [32,33]. Its pleiotropic effects are mediated through the interaction with two receptors: TNFR1 signaling appears to
be involved in the induction of neuroinflammatory processes, while TNFR2 engages in cell survival neuroprotection and sustains homeostasis processes. The balance

Page 8 of 14

between TNFR1 and TNFR2 levels and their activation
status determine the complexity of TNF-TNFRs pathways. Alterations in the TNFR1-TNFR2 balance havebeen confirmed in MS [53] in association with a more
severe and early disease progression [38]. Transgenic
EAE mouse models have contributed significantly tounderstanding the pathological role of TNF and TNFRs
in MS [49]. TNF/TNFR1 KO mice exhibited a milder disease course [68, 69], whereas TNFR2 KO mice showed
more severe EAE symptoms and diffuse demyelination[69]. Specifically, the selective ablation of TNFR2 in oligodendrocytes results in impaired remyelination [83].
Likewise, TNFR1 signaling activation by TNF mediates
necroptosis and apoptosis in oligodendrocytes [35, 36]
and causes neuronal excitotoxicity [78] and necroptosis in cortical neurons [81]. These processes contributeto inflammatory demyelinating processes and neurodegeneration. Therefore, it is unsurprising that TNF and
TNFR1 are overexpressed in CALs and GM lesions in
MS patients [30, 38, 95, 99], especially in those with progressive MS [38]. In addition, increased levels of TNF
were detected in the CSF of MS patients [26]. High CSF
TNF levels are associated with chronic compartmentalized inflammation, causing GM damage from the early
disease stages, correlating with the degree of disability inpatients with progressive MS [20, 26, 28, 29, 84, 85]. All
these results (summarized in Table 1) suggest a possible
role for TNF-TNFR1 signaling activation in disease progression independent of acute inflammation and in thedecrease of compensation mechanisms following a neuronal insult [20, 28, 87, 88].
Neurodegenerative processes drive progression independent of relapse activity, PIRA, which is currently
considered the main contributor to irreversible disability accumulation since the relapse-onset of the disease
and throughout the entire disease course [9, 14, 15, 116,117]. PIRA plays a significant role in worsening and transitioning to progressive MS. However, the definition of
PIRA is not widely clarified as disease progression can be
extremely gradual and slow compared to relapses. This
makes PIRA underestimated and not so easily recognized[118]. Furthermore, consistent data have shown no beneficial effects of DMT on PIRA [117, 124, 125].
Similarly, TNF-TNFRs blockers used for the treatment
of several inflammatory diseases are not only ineffective
but also potentially harmful for MS patients [66, 128].
Their unselective action fails to preserve neuroprotective and cell survival processes associated with TNFR2
signaling.
For these reasons, understanding PIRA is crucial fordeveloping effective MS therapies. Selective modulators targeting TNFRs (e.g., TNFR2 activation or TNFR1
silencing) could be promising therapeutic options.

Mazziotti et al. Journal of Neuroinflammation(2024) 21:209

Page 9 of 14

Table 1 Studies on the role of TNF in MS and EAEType Cells
Involved
MS patients TNF in the MS CSF Macrophages, T
cells

Effects

Relevance

Relevance in MS

References

Infiltrationof activatedmacrophages/T
cells in the brain
parenchyma

MS lesions formation

Inflammation

Disability accumulation[57–59]Neurons, Glial cells Activation of neu- MS lesions formarons and glial cells tion

Neuroinflammation

Neurons

Cortical lesions
and Atrophy

GM demage

Neurodegeneration

Microglia, T cells

Chronic active
lesions

WM lesions

Demyelination

Macrophages, T
cells

Infiltrationof activatedmacrophages/T
cells in the brain
parenchyma a

WM lesions

Neuroinflammation

Diffuse Demyelination[60]

Neurons

AMPAR/NMDAR
Overexpression

Neuronal Excitotoxicity

Neurodegeneration

Severe Disease
Symptoms[78]

EAE TNF KO mice

Macrophages,
T cells, Neurons,
Glial cells

Infiltration
reductionby macrophages
and T cells; No
AMPAR/NMDAR
Overexpression;
Enhanced Tregs
cells

Neuronal homeosasis

Neuroprotection

Severe Disease
Symptoms[68]

EAE TNFR1 KO
mice

Macrophages,
T cells, Neurons,
Glial cells

No AMPAR/NMDAR OverexpressionEnhanced
Tregs cells

Remyelination
Neuroprotection
Increase and Neurodegeneration
Reduction

Reductionof disease signs
and clinical symptoms[69]

EAE TNFR2 KOmice

T regsImpaired Remyeli- Demyelination
Suppressionof ­Tregs in response nation
to autoreactive
T cells

Aggressive
Disease[75, 76]

Oligodendrocytes

Oligodendrocytes
Death

Severe Disease
Symptoms;
Enhanced T
cells infiltrationin the CNS; Diffuse
Demyelination[69, 83]

TNF in MS Lesions

EAE model

Effects

EAE mice

Therefore, the detection of TNF and its receptors serum
levels may be useful in assessing the pharmacological efficacy of DMTs in PIRA.

Conclusions and perspectives
The impact of TNF on MS involves intricate molecular
processes, and understanding these mechanisms is essential for improving treatment strategies and assessment
for PIRA. Specifically, a receptor-selective modulationof the TNF signal pathway could provide a novel therapeutic strategy to attenuate the progression of disability
independent of relapse activity in RRMS. However, this
topic requires further research to fully grasp the potential

Impaired Remyeli- Demyelination
nation[26, 29, 84,
85]
Disability progression

[38]
[99]

therapeutic implications. This promising field of study
could enhance the quality of life for people with MS.
Abbreviations
TNF	Tumor necrosis factor
MS	Multiple sclerosis
PIRA	Progression independent of relapse activity
CNS	Central nervous system
RRMS	Relapsing–remitting MS
SPMS	Secondary progressive MS
PPMS	Primary progressive MS
PrMS	Progressive MS
GM	Grey matter
EDSS	Expanded disability status scale
WM	White matter
CALs	Chronic active lesions
CSF	Cerebrospinal fluid
TNFR	TNF receptor
TNFR1	TNF receptor type-1

Mazziotti et al. Journal of Neuroinflammation(2024) 21:209

TNFR2	TNF receptor type-2
TACE	TNF converting enzyme
mTNF	Transmembrane TNF
sTNF	Soluble TNF
Th	T helper
TRADD	TNFR1-associated death domain
FADD	Fas-associated death domain
RIPK	Receptor-interacting serine/threonine-protein kinase
MLKL	Mixed lineage kinase domain-like pseudokinase
TRAF2	TNFR-associated factor 2
JNK	C-Jun-N-terminal kinase
ERK	Extracellular signal-regulated kinase
NFκB	Transcription factor nuclear factor-κB
EAE	Experimental autoimmune encephalomyelitis
WT	Wild typeKO	Knockout
AMPA	α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
NMDA	N-methyl-D-aspartate
DMTs	Disease-modifying treatments
sNfLs	Serum neurofilament light chainGA	Glatiramer acetate
DC	Dendritic cell
Author contributions
Conceptualization, V.M. and M.C.; writing—original draft preparation, V.M.;
writing—review and editing, F.C.; visualization, E.T.; M.G.; M.B.; S.Z.; F.V.; V.C.;
D.M.; A.T.; supervision, M.C. All authors have read and agreed to the publishedversion of the manuscript.
Funding
This work was supported by #NEXTGENERATIONEU (NGEU) and fundedby the Ministry of University and Research (MUR), National Recovery and
Resilience Plan (NRRP), project MNESYS (PE0000006)—A multiscale integrated
approach to the study of the nervous system in health and disease (DN. 1553
11.10.2022).
Availability of data and materials
No datasets were generated or analysed during the current study.

Declarations
Ethics approval and consent to participate
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 7 June 2024 Accepted: 31 July 2024

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