Секс-хромосомное дополнение и Активин-А формируют терапевтический потенциал активации TNFR2 в модели MS и CNP.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder with a patient population of over 1 million in the United States and 8 million globally (Walton et al., 2020). MS exhibits a profound sex-bias with over 3 to 4 times higher prevalence in females, however, disease progression is more rapid and severe in males ( 1 – 5 ). There is no known cure for MS, and over 50% of patients suffer from mobility disorders or paralysis, independent of sex ( 6 , 7 ).

Neuroinflammation is a key driver in MS progression ( 8 , 9 ) and current treatments rely on immunosuppressive disease-modifying therapies (DMTs). However, DMTs offer limited therapeutic efficacy and carry significant adverse effects ( 10 – 13 ), emphasizing the need for the development of more therapeutically efficacious treatments.

Since inflammation and neurodegeneration are prominent features of MS-associated pathology ( 8 , 9 ), targeting endogenous anti-inflammatory and neuroprotective pathways represents a promising therapeutic strategy. One such target is transmembrane tumor necrosis factor (TmTNF)/TNF receptor 2 (TNFR2) signaling and its therapeutic role in neuroinflammatory disorders ( 14 – 19 ). TmTNF preferentially binds to TNFR2, promotes immune suppression and myelination, and confers neuroprotection ( 20 – 23 ). Despite its therapeutic potential, most studies investigating the therapeutic effects of TmTNF/TNFR2 signaling have focused on a single sex, missing the potential sex-specific response to TNFR2 activation ( 15 – 17 , 24 ).

We have previously shown that TNFR2 activation using a novel TNFR2 agonist in experimental autoimmune encephalomyelitis (EAE, a rodent model of MS), alleviates neuropathic pain in male and female mice, yet improves motor function only in females. Moreover, TNFR2 knock-out (TNFR2-KO) females had exacerbated motor deficits compared to wild-type (WT) controls and no differences were observed between TNFR2-KO and WT males ( 18 ). These data demonstrated the sex-specific attenuation of motor deficits via TNFR2 activation in EAE females.

The purpose of this study was to investigate male sex-specific mechanisms that may limit EAE motor recovery despite TNFR2 activation. Utilizing sophisticated pharmacology, four-core genotype transgenic mice, and gonadectomized mice, we sought to identify the male-specific sex-characteristics that may account for the limited therapeutic efficacy of TNFR2 activation in EAE males.

Results

TNFR2 Activation Sex-Independently Alleviates Sensory Deficits but not Motor Symptoms.

Previous studies have demonstrated that TNFR2 activation alleviated EAE-induced neuropathic pain (measured by evoked pain-response) in both sexes; however, motor disease attenuation was selective in females ( 18 ). In the present study, we show that administering three doses of our next-generation TNFR2 agonist significantly attenuates not only evoked (von Frey), but nonevoked (MCA) pain responses as well, in a sex-independent manner ( Fig. 1 B – E ). Open field testing for motor deficits demonstrated a significant delay in motor symptom onset and reduced disease severity, represented as a cumulative disease index (CDI), following TNFR2 activation until DPI 30 ( Fig. 1 F and H and Table 1 ) in both sexes. However, at DPI 40, therapeutic efficacy for attenuation of motor disease was sustained only in females, and males had diminished therapeutic efficacy for motor symptoms between DPI 30 and 40 ( Table 1 ). Survival analyses demonstrated that TNFR2 activation significantly increased the proportion of female mice that do not develop paresis, with no paresis modifying effect in male mice ( Fig. 1 G and I ). The observed changes in motor disease development and severity were not influenced by additional TNFR2 agonist administration since a fourth supplemental dose of the TNFR2 agonist administered at DPI 24 had no effect on disease outcomes following EAE induction in males ( SI Appendix , Fig. S1 and
Table S2 ).

TNFR2 activation alleviates mechanical hypersensitivity in both sexes but alleviation of motor disease severity is sex-dependent in EAE. Male and female WT mice were immunized to develop EAE and at DPI 10, 13, and 16, they were administered with either TNFR2 agonist (10 mg/kg) or with PBS, indicated in black arrows. ( A ) Timeline of the experiment (created through Biorender.com). Sensory deficits were analyzed for females and males using ( B and C ) von Frey ( B , n = 20 each; C , n = 10 each) and ( D and E ) MCA (n = 5 each). ( F ) Clinical representation of EAE females who developed motor deficits (WT females + PBS, n = 13; WT females + TNFR2 agonist, n = 8). ( G ) Survival curve depict the probability of remaining paresis free after TNFR2 agonism in females. ( H ) Clinical representation of EAE males who developed motor deficits (EAE males + PBS n =11; EAE males + TNFR2 agonist n = 15). ( I ) Survival curve depict probability of remaining paresis free after TNFR2 agonism in males. Data are represented as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Table 1. Clinical parameters of motor deficits A B C D EAE females + PBS EAE females + TNFR2 agonist Significance A vs. B EAE males + PBS EAE males + TNFR2 agonist Significance C vs. D Day of onset 18.77 ± 1.05 26.28 ± 2.59 ** 20.55 ± 0.97 29.27 ± 1.44 *** CDI 14-30 38.16 ± 4.05 15.47 ± 5.82 ** 28.66 ± 3.31 8.4 ± 3.10 *** CDI 30-40 27.39 ± 0.81 18.38 ± 1.73 **** 21.32 ± 1.13 19.43 ± 1.13 ns

Clinical parameters of motor deficits

XY Males have Increased Motor Disease Severity Compared to XX Males upon TNFR2 Activation.

Since TNFR2 activation provided transient relief for motor disease severity in males relative to sustained protection in females, we investigated male-specific characteristics that may impede longer therapeutic effects of TNFR2 activation. Previous research has established that XY male sex-chromosome complement is immunostimulatory and contributes to neurodegeneration in EAE models ( 25 ). Therefore, we first examined the contribution of the male sex-chromosome complement (XY) on the limited efficacy of TNFR2 activation by using XX males (XXM/XX sry+ ) from the FCG model, a well-established model for studying sex-chromosome effects ( 26 ). XXM and XY males (XYM) underwent EAE induction and were subsequently treated with the TNFR2 agonist.

TNFR2 agonist–treated XXM and XYM mice have significantly higher withdrawal thresholds compared to vehicle controls ( Fig. 2 A ). Furthermore, agonist-treated XXM mice demonstrate a reduced escape latency compared to vehicle-treated XXM controls ( Fig. 2 B ). There was no observable difference in motor disease onset and progression between vehicle-treated XXM and XYM mice ( Fig. 2 C , D). However, while the agonist delayed motor disease onset in both gonadal males ( Table 2 ), only XXM experienced sustained attenuation of motor disease severity ( Fig. 2 E and Table 2 ). Furthermore, we verified the XY sex-chromosome-dependent limitation in TNFR2 mediated efficacy by examining XY and XX females and found that XY females experienced greater motor disease severity than XX females ( SI Appendix , Fig. S2 ). Survival analyses evaluating the proportion of mice that develop paresis showed that XX males are significantly protected against paresis onset relative to XY males ( Fig. 2 F ). Strikingly, XY males were four times as likely to develop paresis (80%) compared to XX males (20%) following TNFR2 activation.

Motor disease incidence and motor disease severity is significantly reduced in EAE XX sry+ males as compared to EAE XY males upon TNFR2 activation. XY and XX sry+ males (XX males) were immunized with EAE and at DPI 10, 13, and 16, mice were administered with either TNFR2 agonist (10 mg/kg) or with PBS. ( A ) Withdrawal threshold was measured by von Frey for XX and XY males with either PBS or TNFR2 agonist treatment (XY males n = 10 each group; XX males + PBS n = 15; XX males + TNFR2 agonist n = 17). ( B ) Escape latency of XX males after TNFR2 agonist or PBS treatment (n = 5 each group). ( C ) Representation of clinical scores of EAE XX males and EAE XY males who developed motor deficits (XX males + PBS n = 7; XX males + TNFR2 agonist n = 21; XY males + PBS n = 5; XY males + TNFR2 agonist n = 8). ( D ) There is no difference in motor disease presentation in EAE XY and XX males after PBS treatment (n = 7 XX sry+ males + PBS; n = 5 XY males + PBS). ( E ) Clinical representation of difference in motor disease presentation between XX and XY males after TNFR2 agonist treatment (n = 4 XX sry+ males + TNFR2 agonist; n = 8 XY males + TNFR2 agonist). ( F ) Survival curve depict probability of remaining paresis-free after TNFR2 agonism in XX and XY males. Data are represented as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Table 2. Clinical parameters of motor deficits A B C D EAE XY males + PBS EAE XY males + TNFR2 agonist EAE XX males + PBS EAE XX males + TNFR2 agonist Significance Day of onset 21.20 ± 1.98 29.13 ± 1.06 24 ± 2.18 27.5 ± 3.17 A vs. C ns and B vs. D ns CDI 14-30 26.48 ± 2.79 7.844 ± 2.56 22.66 ± 6.077 12.38 ± 4.16 A vs. C ns and B vs. D ns CDI 30-40 24.45 ± 1.62 27.57 ± 0.811 26.57 ± 1.54 18.28 ± 2.94 A vs. C ns and B vs. D **

Clinical parameters of motor deficits

TNFR2 Agonist–Treated XYM Experiences Greater Neurodegeneration Compared to TNFR2 Agonist–Treated XXM and XXF.

Previous work has established the role of tmTNF/TNFR2 pathways in activating anti-inflammatory and neuroprotective responses ( 20 , 21 ). Following EAE induction, TNFR2 activation only provided sustained alleviation of severe motor disease in animals with the XX sex-chromosome complement. This then prompted us to investigate whether TNFR2 activation elicits neuroprotective and anti-inflammatory effects in a sex-chromosome-dependent manner within our model. Naive XX females (XXF), XX males (XXM), and XY males (XYM) underwent EAE induction and were subsequently treated with the TNFR2 agonist or vehicle. At DPI 40, motor cortex samples were analyzed for several pathological markers from animals exhibiting motor deficits.

To examine the presence and severity of demyelination, a hallmark of MS/EAE ( 27 – 29 ), we measured myelin presence using fluoromyelin red stain, which is a commonly used lipid-based stain that stains myelin ( 30 ). Data were normalized to naive myelin expression and we found a significant reduction in fluoromyelin in PBS-treated EAE mice across all groups—XXF, XXM, and XYM ( Fig. 3 A ). Agonist-treated XXF and XXM showed significantly increased fluoromyelin expression compared to their vehicle-treated controls, but no difference was observed in XYM.

TNFR2 activation promotes myelin and axonal repair pathways and reduces inflammatory burden in XX sex-chromosome complement type. Represented pictographs of ( A ) myelin expression, ( B ) number of myelinating oligodendrocytes, ( C ) axonal damage, and ( D ) the number of activated microglia/macrophages by immunohistochemistry in the motor cortex of EAE animals treated with TNFR2 agonist or PBS. n = 4 each group. The scale bar is 50 μm for all the images. Data are represented as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Furthermore, we analyzed opalin expression which is a transmembrane protein specific to actively myelinating oligodendrocytes ( 31 , 32 ). Opalin-positive nuclei (marked with DAPI, Fig. 3 B ) were quantified per mm 2 and opalin counts were significantly higher in agonist-treated EAE XXF and XXM compared to vehicle-treated controls. However, no significant changes were observed in XYM ( Fig. 3 B ).

To further determine the degree of sex-chromosome complement-dependent TNFR2 agonist-mediated neuroprotection following EAE induction, we evaluated changes in SMI312 levels, an axonal marker for healthy neurofilaments that has been previously shown to be significantly reduced in similar EAE models ( 33 ). In line with previously published studies, we found significantly lowered SMI312 expression levels in vehicle-treated EAE male or female mice, and this effect was independent of the sex-chromosome complement ( Fig. 3 C ). However, TNFR2 activation selectively increased SMI312 levels in XXF and XXM, but no change was observed in XYM mice ( Fig. 3 C ).

To evaluate inflammatory response, we quantified reactive microglia/macrophages using IBA1, a calcium-binding protein indicative of proinflammatory status ( 34 , 35 ). IBA1-positive cells were assessed for the presence of reactive microglia (red arrows, Fig. 3 D ), an additional marker indicating a proinflammatory environment ( 35 ). We counted reactive IBA1+/DAPI+ nuclei and found significantly reduced numbers in XXF and XXM after TNFR2 agonist treatment, but no reduction in XYM mice ( Fig. 3 D ).

Testosterone Replacement does not Alter Motor Disease Severity in Gonadectomized Males upon TNFR2 Activation.

Although our findings indicate a robust sex-chromosome-dependent effect, we further investigated the potential role of male sex hormone testosterone since sex chromosomes and hormones may independently limit the TNFR2 activation efficacy for sustained motor recovery. Adult gonadectomized male mice were used to evaluate the specific influence of testosterone on EAE severity and TNFR2 agonist treatment efficacy. Mice were gonadectomized following sexual maturity to mitigate any confounding sex-hormone-dependent developmental influences. Testosterone levels were replaced in a subset of these mice prior to EAE induction and TNFR2 agonist treatment. The clinical progression of motor deficits across all groups is shown ( Fig. 4 B ). However, to identify the role of testosterone in TNFR2 agonism therapeutic efficacy, we performed a focused analysis between gonadectomized EAE males with a placebo pellet and TNFR2 agonism and gonadectomized EAE males with testosterone replacement and TNFR2 agonism ( Fig. 4 C ). Males without testosterone replacement developed earlier onset of motor symptoms compared to those with exogenous testosterone replacement despite TNFR2 agonist treatment. However, no significant difference in motor disease severity was observed between testosterone-replaced and testosterone-absent males following treatment ( Fig. 4 C and Table 3 ).

Testosterone delays motor disease onset and but does not reduce motor disease severity in EAE males. ( A ) Timeline of the experiment (created through Biorender.com). ( B ) Male motor scores that reflect the progression of motor impairment in all groups [WT + PBS n = 5; WT + TNFR2 Agonist n = 8; Gonadectomized (placebo + PBS) n = 14; Gonadectomized (placebo + TNFR2 Agonist) n = 13; Gonadectomized (testosterone + PBS) n = 3; Gonadectomized (testosterone + TNFR2 Agonist) n = 7]. ( C ) Male motor scores that reflect the progression of motor impairment for TNFR2 agonist-treated gonadectomized males receiving either placebo or testosterone replacement [Gonadectomized (placebo + TNFR2 Agonist) n = 13; Gonadectomized (testosterone + TNFR2 Agonist) n = 7]. ( D ) Day of motor disease onset for the gonadectomized EAE males receiving either placebo or testosterone replacement. Data are represented as mean ± SEM; ** P < 0.01.

Table 3. Clinical parameters of motor deficits Gonadectomized (Placebo + TNFR2 agonist) Gonadectomized (testosterone + TNFR2 agonist) Significance Day of onset 25.92 ± 1.17 31.43 ± 0.42 ** CDI 14-30 13.09 ± 3.76 2.98 ± 0.76 * CDI 30-40 29.83 ± 0.68 26.02 ± 1.32 ns

Clinical parameters of motor deficits

TNFR2 Activation does not Attenuate Neuropathic Pain in Gonadectomized Males.

While investigating the role of testosterone in sustained attenuation of motor disease following TNFR2 agonism, we observed that none of the gonadectomized males recovered from neuropathic pain following treatment, even with testosterone replacement ( Fig. 5 A ). Increased withdrawal threshold, as indicative of reduced mechanical hypersensitivity, following TNFR2 agonism in EAE males required intact gonads ( Fig. 5 A ). Following MCA testing, gonadally intact EAE males treated with the TNFR2 agonist demonstrated reduced escape latency compared to controls ( Fig. 5 B ). However, gonadectomized EAE males treated with the TNFR2 agonist had similar escape latencies to gonadectomized PBS-treated EAE males, irrespective of testosterone replacement ( Fig. 5 B ). We tested whether directly altering testosterone levels in gonadally intact male mice using the androgen receptor blocking drug flutamide prior to EAE induction altered TNFR2 agonist treatment efficacy. In line with our hypothesis, we found that flutamide treatment had no impact on ability of TNFR2 agonism to mitigate EAE-induced pain ( Fig. 5 C and D ).

Therapeutic alleviation of CNP by TNFR2 activation is male gonad dependent. ( A ) von Frey analysis for gonadally intact and gonadectomized males (n = 10, WT EAE males + PBS and TNFR2 agonist each; n = 18 EAE gonadectomized males in each group). ( B ) Escape latency was analyzed by MCA for all the groups (n = 5 each). Gonadally intact males were either implanted with the flutamide (antiandrogens) or a placebo pellet 10 d before EAE immunization. ( C and D ) Effect of Flutamide on withdrawal threshold and escape latency after TNFR2 agonist treatment ( C , n = 20 each; D , n = 5 each). Data are represented as mean ± SEM; * P < 0.05 ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Activin-A Antagonism Blunts TNFR2 Agonist–Mediated Axonal Recovery and Alleviation of Chronic Neuropathic Pain.

Since gonadectomized males never recovered from CNP with TNFR2 activation, we next wanted to identify the bioactive compounds secreted by male gonads necessary for this therapeutic effect. Activins, specifically Activin-A, are produced by male gonads and are primarily involved in spermatogenesis. However, Activin-A has also been linked to inflammation and neuropathic pain ( 36 , 37 ). Follistatin, an Activin-A antagonist, binds to its soluble form and blocks receptor binding plus subsequent activity ( 38 – 40 ). To investigate the role of Activin-A in TNFR2-mediated NP relief in EAE males, gonadally intact males received Follistatin at DPI 9, after NP onset ( 41 ) and prior to TNFR2 agonist treatment. Treatment with both Follistatin and the TNFR2 agonist significantly reduced withdrawal thresholds in EAE males compared to those receiving only the agonist ( Fig. 6 B ). Similarly, Follistatin-treated EAE males showed increased escape latency compared to controls treated with agonist only ( Fig. 6 C ).

Alleviation of CNP by TNFR2 activation is dependent on Activin-A. ( A ) Timeline of the Follistatin experiment in EAE (created with Biorender.com ). ( B ) von Frey analysis for males administered with Follistatin and TNFR2 agonist (n = 10 EAE males + PBS and EAE males + TNFR2 agonist each; n = 13 EAE males + follistatin + PBS and EAE males + follistatin + TNFR2 agonist each). ( C ) MCA analysis for males administered with Follistatin and TNFR2 agonist (n = 5 each). ( D ) von Frey analysis for females administered with Follistatin and TNFR2 agonist (n = 5 EAE females + PBS and EAE females + TNFR2 agonist each; n = 8 EAE females + follistatin + PBS and EAE + follistatin + TNFR2 agonist each). ( E ) MCA analysis for females administered with Follistatin and TNFR2 agonist (n = 5 each). ( F , G ) Lumbar spinal cords of males suffering from EAE neuropathic pain were analyzed for NF200 expression (n = 4, each group). ( H ) Timeline of the Follistatin experiment in CCI (created with Biorender.com ). ( I , J ) Depicted withdrawal thresholds of males and females with CCI and TNFR2 agonist treatment paired with Follistatin (n = 8 each). Data is represented as mean ± SEM; * P < 0.05 ** P < 0.01, *** P < 0.001, **** P < 0.0001.

We next evaluated whether the influence of Activin-A is sex-independent. Since female gonads also produce Activin-A ( 38 ), we evaluated this using gonadally intact female EAE mice treated with Follistatin and TNFR2 agonist as described above. Our findings in female mice replicate the abolishment of TNFR2 agonist pain relief efficacy in male mice ( Fig. 6 D and E ). Together, these data suggest that pain attenuation in both sexes is Activin-A dependent. Surprisingly, there was no effect of Activin-A inhibition in motor disease onset and its severity after TNFR2 activation in both sexes ( SI Appendix , Fig. S3 A – D ).

Our lab has previously shown that TNFR2 activation enhances axonal recovery in models of peripheral nerve injury associated with CNP ( 19 ). NF200 expression, a marker of healthy axons, was probed in EAE males suffering only from CNP. We found that TNFR2 agonist–treated EAE males showed significantly elevated levels of NF200 in the lumbar spinal cord as compared to when Follistatin was coadministered with the TNFR2 agonist ( Fig. 6 F and G ).

Finally, we wanted to determine whether Activin-A-dependent CNP alleviation by TNFR2 activation is EAE specific or if there is translation to different CNP models. In chronic constriction injury (CCI), a peripheral nerve injury model that elicits neuropathic pain, our lab has previously established that TNFR2 activation alleviates CNP ( 19 ). In this study, when CCI males and females were treated with Follistatin and TNFR2 agonist, they had significantly lowered withdrawal thresholds compared to those treated with the TNFR2 agonist alone ( Fig. 6 I and J ). Altogether, these data indicate that Activin-A is critical in TNFR2-mediated CNP alleviation in both a disease and injury model.

Discussion

TNFR2 activation leads to Treg expansion and enhances immune suppression, positioning TNFR2 as a potential therapeutic target for autoimmune disorders like MS ( 14 , 19 , 20 ). However, few studies have explored its therapeutic efficacy in a sex-specific manner. Previous work by our lab has demonstrated that TNFR2 activation alleviates neuropathic pain in both male and female EAE mice but attenuates motor symptoms only in females ( 18 ). Here, we dissected the role of the male sex-chromosome complement and sex hormones to identify the male sex-specific characteristic that limit TNFR2 agonist alterations in male motor disease severity. We found that this change in TNFR2 agonist efficacy is specific to the male XY sex-chromosome and not the male sex-hormone testosterone. Complementing this, we found that the sex-independent effects of TNFR2 agonist decreases in pain are driven through a Activin-A-dependent mechanism, and this mechanism of action can be generalized to other pain models.

The Contribution of Sex-chromosome and Testosterone in TNFR2-Mediated Motor Recovery.

Previous research using whole TNFR2-KO mice supports a female bias in TNFR2-mediated neuroprotection ( 18 ). Supporting evidence from both human and animal studies demonstrates that relative to female, males with MS (humans) or EAE (mice) exhibit higher levels of neurodegeneration and increased disease severity ( 42 – 44 ). This may indicate that females possess a more robust endogenous TNFR2-dependent protective mechanism which leads to enhanced disease resilience.

The XX sex-chromosome complement in females could contribute to this resilience. The second X chromosome has been implicated in neuroprotection in MS and other neurodegenerative diseases such as AD ( 45 , 46 ). This protective effect may stem from the suppression of TLR7 expression in a XX sex-chromosome complement type due to parental imprinting ( 46 ).

Increased TLR7 expression in XY males has been shown to exacerbate EAE severity by downregulating FoxP3+ regulatory T-cells, a critical immune population required for TNFR2-mediated anti-inflammatory effects ( 19 , 47 , 48 , 49 ). Reduced regulatory T cell levels may limit their expansion upon TNFR2 activation and could contribute to the limited therapeutic effects observed in EAE XY males. Prior work by Du et al. supports a role for TLR7 in modulating immune responses, it is important to note that the FCG model used in this study harbors a recently documented X–Y translocation encompassing the TLR7 gene ( 50 ).

We accounted for potential confounding effects associated with X–Y immune–related gene translocations in our studies by using WT XY males as controls. In doing so, we are able to ensure that phenotypes observed in our study cannot be attributed to X–Y translocation–related increases in X-linked TLR7 or other immune-related gene expression as has been previously noted for this FCG mouse model ( 50 ).

Our findings were further validated by comparing the effect of TNFR2 agonism on motor disease severity in FCG model derived XY-female when compared to WT XX female mice ( SI Appendix , Fig. S2 ). We show that EAE induction in XY females similarly phenocopies our findings in WT XY males, regardless of TNFR2 agonist treatment. While the documented X–Y translocation in the FCG model was demonstrated to increase the expression of X-linked immune related genes such as TLR7, this did not have an influence on the behavioral outcomes observed in our experimental model. The similarity in disease phenotypes between XY females and WT XY males would suggest that, in the context of our experiments, the translocation does not impact the immune mechanisms underlying findings. The observed increase in XY - female motor disease severity relative to XX females provides further evidence for an XY-mediated limitation of TNFR2 agonist therapeutic efficacy in EAE.

Our findings provide significant insight into the role of sex chromosomes in mediating EAE disease severity and provide rationale for the pursuit of additional therapeutic strategies aimed at addressing limitations in the therapeutic efficacy of TNFR2 activation observed in XY males. TLR7 suppression may blunt disease exacerbation by limiting the downregulation of FoxP3+ regulatory T cells and subsequently mitigating proinflammatory signaling. As such, inhibiting TLR7 ( 51 ) in conjunction with TNFR2 activation may work to augment therapeutic efficacy in EAE males with XY sex-chromosome complement.

Testosterone plays a significant role in conjunction with TNFR2 activation by delaying the onset of motor symptoms in EAE. Testosterone’s immunosuppressive properties, including suppression of soluble TNF levels and reduction of oxidative and nitrosative stress, are well documented ( 25 ). These mechanisms may account for the efficacy of testosterone replacement therapy in clinical trials for males with MS ( 52 , 53 ).

Our findings reveal a fourfold decrease in the proportion of XX males that remain paresis-free following EAE induction and TNFR2 activation compared to XY males. Furthermore, this reduction in motor disease incidence upon TNFR2 activation was more pronounced in XX males than in XX females (compared to their PBS treated counterparts, SI Appendix , Table S1 ), with a 20% greater protective effect in XX males. This difference reflects the combined benefits of the XX chromosome complement and testosterone-mediated delayed motor disease onset, providing XX males with a distinct advantage over both XY males and XX females.

These findings underscore the importance of sex-chromosome-informed approaches to MS treatment. TNFR2 activation shows significant promise for limiting disease progression in females but warrants further investigation in males due to the limitations posed by the XY chromosome complement.

TNFR2 Activation in Chronic Neuropathic Pain: Activin-A Dependency.

Chronic neuropathic pain (CNP) is highly prevalent in MS patients impacting 50 to 86% of patients ( 54 ). CNP also more broadly influences approximately 10% of the global population ( 55 ). The resistance to available pain therapies in these populations represents a major unmet medical need. Activation of the TNFR2 pathway promotes peripheral regulatory T cell expansion which reduces inflammation, immune cell infiltration in the CNS, and promotes axonal regeneration, which has been shown to resolve CNP ( 19 ).

Considering the increasing incidence of CNP in elderly population, it is promising that alleviation of CNP by TNFR2 activation is testosterone independent, as the testosterone level declines with age ( 56 – 58 ). Interestingly, alleviation of CNP by TNFR2 activation is dependent on Activin-A. Activin-A is a member of Activins family and is produced by male and female gonads. The primary role of Activin is to stimulate FSH release. However, multiple functions of Activins have now been found, in part due to their widespread types (A–E) and receptor distribution. They are now known to play role in embryonic development, tissue differentiation, energy metabolism, neural development, and inflammation ( 38 ).

While TNFR2 signaling pathway has been extensively studied ( 23 ), its interaction with Activin-A has not been explored, especially in the context of CNP. Our data show that inhibition of Activin-A across multiple pain models, whether EAE or CCI, abolishes the therapeutic effects of TNFR2 activation on CNP. This finding reveals a previously unstudied mechanism. Notably, as Activin-A levels do not decrease with age ( 59 , 60 ); this mechanism remains viable for alleviating CNP in the aging human population, positioning TNFR2 activation as a promising therapeutic strategy for treating chronic pain in conditions like MS.

Our study uncovers multiple sex- and sex-chromosome-dependent mechanisms dictating the efficacy of TNFR2 activation in mitigating neuropathic pain and motor disease in an EAE model. These findings may prove to be translationally relevant to a multitude of autoimmune disorders in humans including MS. We show TNFR2 activation–mediated reductions in motor disease severity is influenced by the sex-chromosome complement and not sex hormones. Particularly, we show that XY chromosome limits TNFR2 agonist therapeutic benefits. The observed resilience of the XX chromosome complement provides insights into sex-based responses, since we showed that XX males experienced reduced disease incidence and severity compared to XY males. Additionally, we identified a role for gonadal Activin-A in mediating CNP through TNFR2 activation, providing a promising avenue for pain-relief interventions that are effective in an aging population.

Material and Methods

Detailed methods can be found in SI appendix material and methods. Briefly, EAE was immunized in both sexes in C57BL/6. Four core genotype mice were on BL/6 background were brought from the Jackson Laboratories. The TNFR2 agonist was administered at DPI 10, 13, and 16. Animal care and treatment were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the George Washington University. Male and female mice were analyzed separately.

Supplementary Material

Appendix 01 (PDF)