Gober et al. Journal of Neuroinflammation(2024) 21:149
https://doi.org/10.1186/s12974-024-03129-0

Journal of Neuroinflammation

Open Access

RESEARCH

Exploratory assessment of the effectof systemic administration of soluble
glycoprotein 130 on cognitive performanceand chemokine levels in a mouse modelof experimental traumatic brain injury
Ian G. Gober1,2, Ashley L. Russell1,2, Tyler J. Shick1,2, Vincent A. Vagni2,3, Jenna C. Carlson5,6, Patrick M. Kochanek2,3 andAmy K. Wagner1,2,4,7,8*
Abstract
Uncontrolled neuroinflammation mediates traumatic brain injury (TBI) pathology and impairs recovery. Interleukin-6(IL-6), a pleiotropic inflammatory regulator, is associated with poor clinical TBI outcomes. IL-6 operates via classicalsignaling through membrane-bound IL-6 receptor (IL-6R) and trans-signaling through soluble IL-6 receptor (s)IL-6R. IL-6 trans-signaling specifically contributes to neuropathology, making it a potential precision therapeutic TBI
target. Soluble glycoprotein 130 (sgp130) prevents IL-6 trans-signaling, sparing classical signaling, thus is a possible
treatment. Mice received either controlled cortical impact (CCI) (6.0 ± 0.2 m/s; 2 mm; 50-60ms) or sham procedures.
Vehicle (VEH) or sgp130-Fc was subcutaneously administered to sham (VEH or 1 µg) and CCI (VEH, 0.25 µg or 1 µg)
mice on days 1, 4, 7, 10 and 13 post-surgery to assess effects on cognition [Morris Water Maze (MWM)] andipsilateral hemisphere IL-6 related biomarkers (day 21 post-surgery). CCI + sgp130-Fc groups (0.25 µg and 1 µg)
were combined for analysis given similar behavior/biomarker outcomes. CCI + VEH mice had longer latencies andpath lengths to the platform and increased peripheral zone time versus Sham + VEH and Sham + sgp130-Fc mice,suggesting injury-induced impairments in learning and anxiety. CCI + sgp130-Fc mice had shorter platform latencies
and path lengths and had decreased peripheral zone time, indicating a therapeutic benefit of sgp130-Fc after
injury on learning and anxiety. Interestingly, Sham + sgp130-Fc mice had shorter platform latencies, path lengths
and peripheral zone times than Sham + VEH mice, suggesting a beneficial effect of sgp130-Fc, independent of
injury. CCI + VEH mice had increased brain IL-6 and decreased sgp130 levels versus Sham + VEH and Sham + sgp130Fc mice. There was no treatment effect on IL-6, sIL6-R or sgp130 in Sham + VEH versus Sham + sgp130-Fc mice.
There was also no treatment effect on IL-6 in CCI + VEH versus CCI + sgp130-Fc mice. However, CCI + sgp130-Fc
mice had increased sIL-6R and sgp130 versus CCI + VEH mice, demonstrating sgp130-Fc treatment effects on brain
biomarkers. Inflammatory chemokines (MIP-1β, IP-10, MIG) were increased in CCI + VEH mice versus Sham + VEH and*Correspondence:
Amy K. Wagner
wagnerak@upmc.edu
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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in this article, unless otherwise stated in a credit line to the data.

Gober et al. Journal of Neuroinflammation(2024) 21:149

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Sham + sgp130-Fc mice. However, CCI + sgp130-Fc mice had decreased chemokine levels versus CCI + VEH mice. IL-6
positively correlated, while sgp130 negatively correlated, with chemokine levels. Overall, we found that systemic
sgp130-Fc treatment after CCI improved learning, decreased anxiety and reduced CCI-induced brain chemokines.
Future studies will explore sex-specific dosing and treatment mechanisms for sgp130-Fc therapy.
Keywords Traumatic brain injury, Interleukin-6, Trans-signaling, Soluble glycoprotein-130, Morris water maze,
Cognition, Anxiety, Chemokines

Introduction
There are no disease-modifying therapies for traumaticbrain injury (TBI) to improve neurologic recovery. TBI
survivors often have lasting cognitive, behavioral, andsensory deficits, which may be linked to chronic uncontrolled inflammation. Following TBI, the pleiotropic
cytokine, interleukin-6 (IL-6), is upregulated acutely andchronically (3-months) [1]. Patients with higher cerebrospinal fluid (CSF) IL-6 levels had worse 6-month global
outcomes following severe TBI. Other reports demonstrate that patients with severe TBI and with higher ratios
at 3 months post-injury of serum IL-6 to the anti-inflammatory cytokine IL-10 also had increased odds of worse
6-month global outcomes [2]. IL-6 contributes to otherinflammatory pathologies like rheumatoid arthritis (RA),multiple sclerosis (MS), asthma, cancer, metabolic syndrome, type 2 diabetes, and inflammatory bowel disease(IBD) [3–5]. Associations with chronic disease states andpoor neurologic outcomes after TBI highlight IL-6 signaling as a possible therapeutic target.
IL-6 also regulates homeostatic and anti-inflammatory
processes [6] and mechanistically acts via both classical and trans-signaling. Classical signaling occurs when
IL-6 binds to membrane-bound IL-6 receptor (mIL-6R)
in a complex with membrane-bound glycoprotein-130(gp130). Classical signaling only occurs in cell types containing mIL-6R, which include microglia, neutrophils,naïve T-cells, and hepatocytes [7, 8]. Also, mIL-6R activation is critically involved in microglial priming acrossthe lifespan by enhancing both pro-inflammatory genes
and major histocompatibility complex (MHC)-II expression [9]. Activating the classical signaling pathway primarily regulates metabolic and regenerative processes[6]. Conversely, trans-signaling involves soluble IL-6R(sIL-6R), which binds IL-6 before binding to the ubiquitously expressed gp130. Soluble (s)gp130 is the natural
inhibitor of the agonistic IL-6/sIL-6R complex acting
as a decoy receptor [10]. IL-6 has three binding sites forreceptors, with site I recognizing IL-6R and sites II and
III recognizing gp130. IL-6 and IL-6R antibodies target
site I of the IL-6/IL-6R complex which affects all (classical, trans) IL-6 signaling. The fusion protein sgp130(-Fc)
is unique in that it selectively targets sites II and III, only
affecting trans-signaling and sparing classical signaling[11]. Despite the differences in upstream signaling mechanisms between classical and trans-signaling, which relyon cell-type specific differences in IL-6 receptor subtype/subunit expression, the intracellular Janus kinsase/signaltransducers and activators of transcription (JAK/STAT3)
signaling does not differ as a function of IL-6 mediatedclassical versus trans-signaling [12]. However, the different effects observed with trans-signaling are a function of
the wide ranging cell types in which signaling occurs aswell as stronger signaling observed compared to classicalpathways [13]. For example, while classical IL-6 signaling is less able to strongly activate STAT3 in endothelial
cells, classical signaling patterns promote cell survival,
while trans-signaling patterns in endothelial cells canpromote inflammatory activation [14]. Relevant to TBI,
research also suggests that IL-6 trans-signaling involving
endothelial cells facilitates intercellular adhesion molecule (ICAM) and monocyte chemoattractant protein(MCP)-1 secretion, while classical IL-6 signaling facilitates IL-8 mediated immune cell transmigration [14].
Broadly speaking, IL-6 trans-signaling propagates inflammation in the central nervous system (CNS), facilitating
microgliosis and astrocytosis through chemokine signaling, contributing to sickness behaviors and cognitive deficits in mice, and increasing risk for neurodegenerative
disorders like Alzheimer’s disease [7, 15–19].
IL-6 has potential as a therapeutic target for inflammatory diseases. For example, IL-6 deficient mice havedecreased susceptibility to experimental autoimmune
encephalomyelitis [4]. Also, anti-IL-6 antibody treatment (Tocilizumab) decreases symptomology in conditions such as RA, and COVID-19 [20, 21]. While provento be beneficial, IL-6 pan-inhibition by anti-IL-6 antibody
treatment affects both trans- and classical signaling causing immunosuppression. sgp130 selectively neutralizessIL-6R, and thus, prevents trans-signaling, while maintaining the beneficial functions of classical signaling.
Importantly, recent studies suggest that treatment witha sgp130 fusion protein (sgp130-Fc) can promote remission for arthritis and IBD [3, 22, 23].
Despite these findings in lipopolysaccharide (LPS)induced inflammation models, in vitro endothelial cell
models, as well as in infectious and systemic autoimmune
diseases, there is limited literature regarding the translational potential of inhibiting IL-6 trans-signaling inacquired brain injury, including TBI. Therefore, we aimed
to determine if selectively inhibiting IL-6 trans-signaling
via systemic sgp130-Fc administration would promoteGober et al. Journal of Neuroinflammation(2024) 21:149

behavioral and neuroinflammatory benefits in a mousemodel of severe TBI.

Materials and methods
Animals

Adult (12–15 weeks) male C57BL/6J mice (N = 75; Jackson Laboratory) were housed in a temperature-, humidity-, and lighting-controlled environment with ad libitum
access to food and water. Mice were randomly assigned
to one of five groups: (1) Sham + Vehicle (Sham + VEH;n = 21), (2) Sham + 1 µg sgp130-Fc (Sham + 1 µg; n = 8),(3) controlled cortical impact (CCI) + VEH (n = 26),
(4) CCI + 0.25 µg sgp130-Fc (CCI + 0.25 µg; n = 10), (5)CCI + 1 µg sgp130-Fc (CCI + 1 µg; n = 10). Mice in the
CCI + 0.25 µg and CCI + 1 µg sgp130-Fc groups had
similar treatment-associated behavioral and brain IL-6
related chemokine outcomes (Supplemental Figs. 2, 3,
and 4), and therefore, were combined for all analyses(CCI + sgp130-Fc) presented in the main text. Supplemental Figs. 2, 3 and 4 present comparative findings
for the CCI + 0.25 µg and CCI + 1 µg outcomes. A study
design chart is provided in Fig. 1.
Mice received subcutaneous administration of either
recombinant mouse sgp130-Fc chimera protein (R&D
Systems) dissolved in 0.1 M phosphate-buffered saline(PBS) or VEH (PBS) on days (D) 1, 4, 7, 10, and 13 postsurgery. The sgp130-Fc dosing strategy (every 3 days)
was chosen due to the 72-hour half-life of the sgp130-Fc
molecule [24]. The 2 doses (0.25 µg and 1 µg) administered in this exploratory study were based on previous
work. Given that the use of systemic sgp130-Fc for TBI isnovel, doses were chosen to fall within the effective dosing range in other published mouse studies identified atstudy inception [25–27].
Controlled cortical impact (CCI)

CCI mice were anesthetized with 4% isoflurane (induction) and 1–2% (maintenance) in 2:1 nitrous oxide/oxygen [28, 29]. A 5 mm craniotomy was made over the leftparietotemporal cortex. Temperature was maintained at
37 ± 0.5ºC (rectal) during surgery. CCI was performed
using a 3 mm flat-tip pneumatic impactor (velocity

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6.0 ± 0.2 m/s; vertical depth 2 mm; duration 50-60ms).
After impact, the bone flap was replaced, sealed withdental cement, and the incision sutured. Mice were
removed from anesthesia and recovered for 30 minutes(min) with supplemental oxygen. Shams underwent allprocedures except for the CCI. Three mice died, none ofwhich received sgp130-Fc treatment. This injury severity
is accompanied by impairments in motor and cognitive
tasks such as Morris water maze (MWM) learning andmemory as well as a lasting brain inflammatory response[30–32].
Morris water maze (MWM)

MWM testing [28] consisted of two phases: (1) learning
acquisition (D14-18) and (2) visible platform (VP; D19).
Testing was conducted in a circular pool (67 cm diameter, 58 cm deep) filled with water (20–22 °C, 28 cmdeep) in a room surrounded by extra-maze visual cueson the walls. During acquisition, mice underwent four
trials that were randomly assigned to one of four starting locations (north, south, east, west) in the pool. A
9 cm circular platform was submerged 0.5 cm below thewater surface in the southwest quadrant. Mice were given
a maximum of 120 s (sec) to locate the submerged platform per trial. Mice were given 4 min inter-trial intervals
in a 37 °C incubator. For VP testing, the platform waselevated 0.5 cm from the water surface. Mice were randomly assigned to one of the four starting coordinates forfour trials and given a maximum of 120 s per trial. Mice
were given 4 min inter-trial intervals in a 37 °C incubator. Latency and swim tracking were recorded using AnyMaze software (Stoelting Co).
Brain luminex assay

On day 21, mice were anesthetized with 4% isofluraneand sacrificed by decapitation [33, 34]. Brains were
flash-frozen, and stored at -80 °C. The brain hemisphere ipsilateral to the contusion was processed andanalyzed using mouse T-cell (Millipore, MCYTOMAG-70 K-27) and soluble cytokine receptor (Millipore,
MSCRMAG-42 K-12) panels following manufacturer’s
protocols. Tissue was homogenized in T-PER (Thermo

Fig. 1 Experimental design. Mice were randomly assigned to receive either sham or CCI [day (D)0]. Starting on D1, mice received either vehicle (PBS;
Sham and CCI), 0.25 µg sgp130-Fc (CCI) or 1.0 µg sgp130-Fc (Sham and CCI). Mice were treated every 3 days with vehicle or sgp130-Fc. On D14-19 mice
underwent Morris water maze (MWM) testing and on D21 tissue was collected for analysis

Gober et al. Journal of Neuroinflammation(2024) 21:149

Fisher Scientific; 300 mg tissue:1 mL T-PER), and supernatant was removed and diluted in PBS to achieve a final
protein concentration of 6 mg/mL. Samples were assayed
undiluted for the mouse T-cell panel. Samples were run
at a 5 times (x) dilution for the soluble cytokine receptor
panel. Concentrations were measured using a Bio-Rad
LX100 and LX200 microplate readers with Bio-Rad BioPlex manager 6.2 software.
Luminex data were processed and subjected to a linear
transformation. We adopted a trimmed mean (TM) analysis of the fluorescence intensity (FI) to address low andout of range low values through a linearization process
to improve overall standard curve fit and increase theaccuracy of values across the range of the standard curve[35, 36]. The TM process removed the highest and lowest 10% of the measured beads for each analyte for eachsample in each well, reducing random error, and providing a more precise measurement of a given well’s FI. The
TM, FI and concentration for each standard were subject
to log transformation to linearize the data. A third orderpolynomial equation, which best fit the distribution of
the log transformed data, was generated from the standards. New sample concentrations were calculated withthe polynomial equation. Plates were run with intra- andinter-plate controls and indexed to one another using theinter-plate control coefficients. The mean intra-plate andmean inter-plate coefficient of variance were < 15%.
Statistical analyses

Experimenters were blinded to treatment groups. Data
were analyzed using STATA-16 or GraphPad Prism-9.
Values are reported as mean ± standard error of the mean
(SEM). Data were tested for distribution and variance.
We used a mixed effects regression model to address therepeated measurements within the MWM assay. Mixed
regression modeling is a flexible analytic approach that
handles correlated data by accounting for the correlation patterns between repeated measurements withineach individual to be explicitly modeled. The flexibility
to accommodate both fixed effects and random effects,
factors whose effects that are assumed to be the same orvaried across individuals respectively, effectively captures
temporal dynamics of the system to address clinically
relevant questions. Within the MWM assay, a random
intercept mixed effects regression model was used togenerate separate intercepts for each mouse to model
within-mouse variation in test performance over time.
This approach incorporates mouse-to-mouse variability in the inference-making process to describe the relationship between the fixed effects and test performance.
We fit models that evaluated the main effects of group,
time, and the group-by-time interaction effect. We thenestimated marginal means on each of MWM acquisition (D14-18) to assess the day-specific effect of group.

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Marginal mean contrasts with Sidak p-value correction were used to perform the post hoc comparisons bygroup. Speed adjusted mixed models were also generated (Fig. 2 and Supplemental Fig. 2) and only differedfrom the unadjusted models (Supplemental Fig. 1 andSupplemental Fig. 3, respectively) in that mean speed wasincluded as a covariate to predict speed adjusted values
for latency, path length and peripheral zone time. Linear
regression was used to compare group relationships on
VP.
Inflammatory markers were compared across groups
using Kruskal-Wallis tests. Multiple comparisons adjustments were applied using Dunn’s Test. All biomarker correlations were performed with Spearman’s (r).

Results
Morris water maze
Speed-adjusted MWM outcomes

There was a main effect of group for mean swim speed(p < 0.001). In general, CCI mice had slower swim speeds
than Sham + VEH mice across all MWM testing days
(p < 0.05; Fig. 2A). To minimize the impact of the varying swim speeds on MWM outcomes, all MWM variables (latency, path length and peripheral zone time)
were adjusted for speed to observe group-specific effects(detailed description in Methods). Group stratified correlations between swim speed and other MWM variables(D14-18) showed group differences in how swim speed
affected latency, path length and peripheral zone time.
Overall, latency, peripheral zone time and path length
were correlated with swim speed (Table 1). These groupdifferences provided the rationale to control for swim
speed as shown in Fig. 1 and Supplemental Fig. 2 whenevaluating other MWM metrics such as latency andperipheral zone time.
Table 1.
We determined the effect of sgp130-Fc treatment after
CCI on MWM learning acquisition (latency, path length)
and anxiety-like (peripheral zone time [37]) behaviors(D14-19), while adjusting for swimming speed. There wasa significant group effect for latency (p < 0.0001), path
length (p < 0.05), and peripheral zone time (p < 0.0001).
CCI + VEH mice had increased latencies to platform versus Sham + VEH mice (D14-17; p < 0.05). CCI + sgp130-Fc
mice had shorter latencies versus CCI + VEH mice [D14(p = 0.076); D15 (p < 0.05); Fig. 2B]. The time spent in the
peripheral zone was greater for CCI + VEH mice versus
Sham + VEH mice (D15-18; p < 0.05) and CCI + sgp130-Fc
mice (D14-17; p < 0.05; Fig. 2C). Sham + sgp130-Fc micespent less time in the peripheral zone versus Sham + VEH
mice (D14; Fig. 2C). CCI + VEH mice had increased path
lengths to the hidden platform versus Sham + VEH (D1417; p < 0.05) and CCI + sgp130-Fc mice (D14-15; p < 0.05;
Fig. 2D). Speed adjusted VP performance is presented inGober et al. Journal of Neuroinflammation(2024) 21:149

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Fig. 2 sgp130-Fc treatment post-CCI on MWM metrics during learning acquisition (D14-18) and visible platform (VP; D19). (a) Mean speed (b) speed adjusted escape latencies (c) speed adjusted peripheral zone time and (d) speed adjusted path length. Acquisition data were analyzed via mixed modelingfor main effect and Sidak post-hoc testing. VP was analyzed using linear regression. Black arrow on D13 represents final sgp130-Fc or VEH administration.
Lines/bars represent mean ± SEM. Significant comparisons (p < 0.05) include: *Sham + VEH vs. CCI + VEH, %Sham + VEH vs. CCI + sgp130-Fc, ^Sham + VEH vs.
Sham + 1 µg sgp130-Fc, #CCI + VEH vs. CCI + sgp130-Fc, +Sham + 1 µg sgp130-Fc vs. CCI + VEH, !Sham + 1 µg sgp130-Fc vs. CCI + sgp130-Fc

Fig. 2B-D. Sham + VEH and CCI + sgp130-Fc mice had ashorter latency to the VP than CCI + VEH mice (p < 0.05;
Fig. 2B). Sham + VEH mice spent less time in the peripheral zone (p < 0.05; Fig. 2C) and had shorter path lengths
to the VP than CCI + VEH mice (p < 0.05; Fig. 2D).
Non-adjusted MWM outcomes

Analysis was also performed on MWM data without
adjusting for variation in swim speed (Supplemental Fig. 1). There was a significant main effect of groupfor acquisition latency (p < 0.0001), peripheral time(p < 0.0001), mean speed (p < 0.0001) and path length(p < 0.05). Multiple comparison findings were similar tothe speed adjusted data presented. Sham + VEH mice(D14-17 and VP; p < 0.05) and CCI + sgp130-Fc mice(D14-15 and VP; p < 0.05) had shorter latencies to thehidden platform than CCI + VEH mice (Supplemental
Fig. 1A). Sham + VEH mice (D15-D18 and VP; p < 0.05)
and CCI + sgp130-Fc mice (D14-17; p < 0.05) spent lesstime in the peripheral zone versus CCI + VEH mice (Supplemental Fig. 1B). CCI + VEH mice had increased path
lengths versus Sham + VEH and CCI + sgp130-Fc mice
(D15; p < 0.05; Supplemental Fig. 1C).
Overall, these data show that CCI impaired learning and memory in the MWM task compared to shamprocedures. Additionally, mice treated with sgp130-Fc
after CCI had improved MWM performance (decreased
latency and path length) compared to injured control(VEH-treated) mice during the testing days most proximate (D14-15) to the last dose of sgp130-Fc. sgp130-Fc
treatment after CCI also reduced anxiety-like behavior

Gober et al. Journal of Neuroinflammation(2024) 21:149

Table 1 MWM variables correlation with mean speed.
Spearman’s (r) correlations for Morris Water Maze variables
correlated with mean speed. Significant comparisons are bolded
and indicate p < 0.05. Italics indicates a trend towards significance(p < 0.1)
TEST GROUP AND VARIABLE
SHAM + VEHICLELATENCY
PATH LENGTH
Peripheral Zone Time
SHAM + SGP130-FC
LATENCY
PATH LENGTH
Peripheral Zone Time
CCI + VEHICLELATENCY
PATH LENGTH
Peripheral Zone Time
CCI + SGP130-FC
LATENCY
PATH LENGTH
Peripheral Zone Time

Spearman r

P-VALUE

0.16280.33850.19370.097
0.00040.04770.51630.65450.69620.0006< 0.0001< 0.0001-0.20080.2032-0.0005

0.022
0.0204
0.99590.09730.44770.3507

0.3357< 0.0001
0.0003(decreased peripheral zone time) compared to injured
controls.

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sgp130-Fc dose response on MWM outcomes

Our initial study design compared the effect of two
doses of sgp130-Fc after CCI (0.25 µg and 1 µg) on outcome. Prior to combining the 0.25 µg and 1 µg sgp130-Fc
groups for Figs. 2 and 3 and Supplemental Fig. 1 analysis,
we assessed our data for group differences in the behavioral and biomarker assays based on treatment dose. No
statistical differences were noted for speed adjusted or
non-adjusted MWM outcomes (latency, peripheral zone
time, path length, mean speed) between the two treatment groups (Supplemental Figs. 2, 3 and 4). There wasa non-significant effect observed for platform latencies(Supplemental Fig. 2B, 3 A) and peripheral zone times(Supplemental Fig. 2C, 3B) in that mice treated with thehigher sgp130-Fc dose (1 µg) had numerically better outcomes than mice treated with the lower sgp130-Fc dose
(0.25 µg).
Neuroinflammation
IL-6 related biomarkers

Brain IL-6, sIL-6R, and sgp130 levels were measured(D21) in the hemisphere ipsilateral to the injury site.
CCI + VEH mice (p < 0.001) and CCI + sgp130-Fc mice(p < 0.05) had increased IL-6 levels versus Sham + VEH

Fig. 3 Brain levels of IL-6 related biomarkers and inflammatory chemokines 21 days post CCI. (a) IL-6, (b) sIL-6R, (c) sgp130, (d) MIG, (e) IP-10, (f) MIP-1β
were analyzed with Kruskal-Wallis tests and Dunn’s Test. Significant comparisons (*) indicate p < 0.05. Bars represent mean ± SEM

Gober et al. Journal of Neuroinflammation(2024) 21:149

mice. sgp130-Fc treatment had no impact on brain
IL-6 levels (Fig. 3A). sIL-6R levels were similar between
CCI + VEH, Sham + VEH and Sham + sgp130-Fc groups;
however, CCI + sgp130-Fc mice had higher sIL-6R levels(p < 0.05) than Sham + VEH mice (Fig. 3B). sgp130 levels
were decreased in CCI + VEH mice versus Sham + VEH
and Sham + sgp130-Fc mice. sgp130 was increased in CCI
and Sham mice treated with sgp130-Fc (CCI + sgp130Fc and Sham + sgp130-Fc) versus CCI + VEH mice(p < 0.0001) (Fig. 3C).
IL-6 related chemokines

The IL-6 associated chemokines, monokine induced by
gamma (MIG, CXCL9), interferon γ-induced protein
10 kDa (IP-10, CXCL10) and macrophage inflammatory
protein-1β (MIP-1β, CCL4) [17, 38] were also measured(D21) in the hemisphere ipsilateral to the contusion [17,38]. Chemokine levels were increased in CCI + VEH
versus shams (p < 0.0001; Fig. 3D-F). sgp130-Fc treatment after CCI (CCI + sgp130-Fc) reduced MIG (p < 0.05;
Fig. 3D), IP-10 (p < 0.05; Fig. 3E), with a trend toward a
reduction in MIP-1β (p = 0.1001; Fig. 3F) compared to
CCI + VEH-treated mice. There were no sgp130-Fc dose
effects (0.25 µg vs. 1 µg) after CCI on IL-6 related biomarkers (Supplemental Fig. 4). Interestingly, though,the higher dose of sgp130-Fc (1 µg) resulted in non-significantly elevated IP-10 and MIP-1β levels comparedto the lower dose of sgp130-Fc (0.25 µg; Supplemental
Fig. 4E-F).
Overall, these data show that sgp130-Fc treatment
increased brain levels of sIL-6R (in CCI + sgp130-Fc)
and increased sgp130 levels (in CCI + sgp130-Fc andSham + sgp130-Fc) on D21. Also, CCI resulted in a persistent increase in IL-6 associated chemokines, and treatment with sgp130-Fc attenuated this injury response.

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IL-6 related biomarkers and chemokine correlations

We evaluated IL-6 family biomarker relationships withchemokines (Spearman r correlations). IL-6 positively
correlated with MIG (R = 0.56, p < 0.0001), IP-10 (R = 0.74,
p < 0.0001), and MIP-1β (R = 0.80, p < 0.0001). sIL-6R also
positively correlated with MIG (R = 0.20, p = 0.089), IP-10(R = 0.28, p = 0.017), and MIP-1β (R = 0.20, p = 0.083),
but to a lesser extent than IL-6. sgp130 negatively correlated with MIG (R=-0.52, p < 0.0001), IP-10 (R=-0.41,
p = 0.0003), and MIP-1β (R=-0.33, p = 0.0037). MIG,
IP-10, and MIP-1β were highly correlated with one
another (Fig. 4). Together, these findings begin to create afoundation for biomarker-based IL-6 readouts, independent of treatment.

Discussion
Neuroinflammation impairs recovery post-TBI. IL-6trans-signaling is a dominant mechanism driving many
forms of CNS pathology making its selective inhibition,
via sgp130-Fc, a promising therapeutic to promote neurorecovery. Similar to clinical TBI studies, our mouse
CCI model showed persistent IL-6 pathology in the setting of neurological deficits [1, 39]. We showed that intermittent systemic treatment with sgp130-Fc after CCI
improved learning and decreased anxiety-like behaviors
in the MWM. Sgp130-Fc treatment also increased brain
sgp130 and sIL-6R and decreased CCI-induced IL-6 family inflammatory chemokine levels when measured 7 days
after the last dose given. Overall, our data suggest significant CNS effects after systemic sgp130-Fc administration in reducing IL-6 associated brain tissue damage andfunctional impairments.
Clinically, IL-6 inhibition promotes anti-inflammatory effects, and inhibitors are used in various disorders
including RA, COVID-19 and cytokine release syndrome[40]. The anti-IL-6R antibody, Tocilizumab, is cardioprotective in cardiac arrest patients. In that single-center

Fig. 4 Biomarker correlation heat map. The data shown are Spearman (r) correlations. * indicates statistical significanceGober et al. Journal of Neuroinflammation(2024) 21:149

trial, Tocilizumab reduced systemic inflammation after
cardiac arrest, evidenced by decreased C-reactive protein
and leukocyte levels [41]. IL-6 signaling inhibition may
also be relevant to TBI pathology. Yang et al., demonstrated that pan-IL-6 inhibition improved TBI-induced
motor deficits and decreased pro-inflammatory cytokines in a mild closed head injury + hypoxia model [42].
However, there are clinical reports showing that longterm use of Tocilizumab is associated with leukoencephalopathy and also worsened depression [43, 44].
Despite some potential promise, the effect of pan-IL-6
inhibition in the CNS after acute brain injury remains
unclear and requires a greater focus on neurologic endpoints [45]. Given the promising results observed with
pan-IL-6 inhibition, selective CNS sIL-6R signaling
blockage after TBI with sgp130-Fc offers therapeuticpotential and the possibility for a reduced side effect profile. Selectively targeting IL-6 trans-signaling via sgp130Fc could preserve regenerative and neurotrophic effects
while reducing immune suppression that is typicallyassociated with suppressed classical IL-6 signaling [23,46].
We found that two different doses of sgp130-Fc similarly improved CCI-induced functional deficits in that
CCI + sgp130-Fc mice had decreased escape latencies
and swim distance (MWM) versus CCI + VEH mice.
These findings suggest that after CCI, systemic sgp130-Fc
administration improves neurorecovery in learning andmemory tasks. However, underlying factors like motivation and anxiety appear to affect how sgp130-Fc influences MWM performance. For example, CCI mice had
slower swim speeds than Sham + VEH mice. Thus, we
adjusted for swim speed variation using a mixed effects
regression model which reduced group variability in
MWM metrics. CCI + sgp130-Fc mice also had decreased
peripheral zone time versus CCI + VEH mice, indicatingthat IL-6 trans-signaling inhibition may influence injuryinduced anxiety-like behaviors. One important relay
for anxiety is the hypothalamic pituitary adrenal (HPA)
axis. HPA axis stimulation via IL-6 contributes to stressand anxiety following TBI [46]. Our data suggest that theeffects of intermittent systemic sgp130-Fc administration
on HPA axis-related pathways and associated behaviors
after TBI require further study.
Uncontrolled neuroinflammation is also associatedwith both anxiety and memory impairments [47]. The
mediolateral (ML) thalamus, a relay to the amygdala, isinvolved in MWM-associated thigmotaxis [48], and CCI
produces lasting microgliosis in the ML thalamus [49].
Sgp130-Fc treatment-associated reductions in inflammatory chemokines may contribute to improvements in
MWM associated anxiety and cognition; however, weevaluated these markers in the hemisphere ipsilateral tothe injury and did not perform measurements in distinct

Page 8 of 12

brain regions. Thus, investigations into cell-type andregion-specific inflammatory patterns associated with
CCI and sgp130-Fc treatment, as well as effector function
targets like the IL-6 downstream JAK/STAT pathway,
are needed to better understand the molecular processes
underlying behavioral improvements [50]. Also, future
work should include additional assays capturing a varietyof cognitive and affective constructs within our modelthat reflect learning and memory as well as anxiety,anhedonia, sociability, and PTSD-like behaviors in orderto further expand our understanding of the potential of
sgp130-Fc as a clinically translatable therapeutic agent to
TBI survivors.
Other work shows that pan-IL-6 inhibition reduced
injury-induced serum IL-6, keratinocyte-derived chemokine, and MIP-1α levels 24 h after hypoxic brain injury inmice [42]. IL-6 neutralization also reduced brain IL-6 levels and serum neuron-specific enolase in that model [42].
Given this work was an exploratory analysis, we chosespecific, readily interpretable inflammatory molecularreadouts and behavioral readouts as primary endpoints.
As such, while we did not directly use histological techniques to measure the extent of neuronal injury after
CCI with/without sgp130-Fc treatment, treatment associated reductions in brain IL-6 related chemokine levels
are suggestive of reduced neuroinflammation after CCI.
However, studies are needed to explore the impact of
sgp130-Fc after TBI on CNS specific biomarker burden
as well as other region-specific histological outcomes.
Systemic sgp130-Fc administration increased brain
sgp130 and sIL-6R, but not IL-6, suggesting that sgp130Fc may effectively enter the brain possibly due to blood
brain barrier disruption and/or other mechanisms liketranscytosis, to directly impact CNS damage. Also, systemic IL-6 signaling may modulate local immune cellchemokine production to facilitate T-cell and macrophage infiltration, which may also impact CNS damage [17, 38, 51]. Moreover, IL-6 also stimulates the HPA
axis and cortisol production, which may have negative
effects on CNS repair and recovery after acute stress andtrauma [52] as evidenced by acute cortisol associations
with cognitive impairment after severe TBI [53]. Thus, inaddition to direct CNS effects, peripheral modulation of
IL-6 production via systemic sgp130-Fc administration
may impact multiple mechanisms of CNS damage, particularly at later time points in our intermittent dosing
regimen when blood brain barrier permeability is not aprominent component of TBI pathology.
Compensatory increases in sIL-6R bioavailability may
be reactionary to the intermittent dosing that ended
seven days prior to tissue collection, however, increased
bioavailability within an appropriately facilitatory local
environment may possibly support recovery mechanisms
such as neurogenesis, wherein neuronal trans-signaling

Gober et al. Journal of Neuroinflammation(2024) 21:149

reportedly has a role [54, 55]. One consideration regarding D21 sIL-6R expression is that the Milliplex assay measures total IL-6R concentrations and cannot differentiate
IL-6 bound sIL-6R versus unbound sIL-6R [56]. Thus,
further evaluation of the bound vs. unbound sIL-6R isneeded. Also, the assay chosen specifically quantified
total sIL-6R, which is likely more impacted by sgp130Fc treatment given its selectivity for trans-signaling [57];however, future work directly comparing membranebound IL-6R levels is warranted [12]. Given that sgp130Fc has a 72-hour half-life in vivo [24], the direct effect on
IL-6 associated biomarkers is unknown. However, intermittent systemic sgp130-Fc dosing reversed CCI-induced
increases in IL-6 sensitive chemokines MIG, IP-10, andMIP-1β, which are known to perpetuate neuroinflammation in other neurological diseases like encephalitis, MS,
and Alzheimer’s disease [17], where IL-6 signaling also
plays a role. IL-6 associated inflammation may impact
recovery and susceptibility to secondary conditions postTBI, including cognitive performance deficits, which arereported in our clinical population [39].
The beneficial effects of sgp130-Fc did not appear to be
restricted to only after CCI. Interestingly, we found thatsham mice treated with sgp130-Fc (Sham + sgp130-Fc)
performed better in the MWM (lower latencies, peripheral zone time, and path length) than VEH-treated shams(Sham + VEH). We administered the highest dose tested
in this study (1 µg) to sham animals with no adverse
effects. This finding may be due to a CNS response tosystemic sgp130-Fc treatment, as demonstrated by theincreased sgp130 levels in sgp130-Fc treated shams.
However, sgp130-Fc treatment in sham mice, did notfurther reduce IL-6 associated chemokine (MIG, IP-10,
MIP-1β). Sham procedures in mice, specifically craniotomy are often associated with some level of brain injury,
and thus these findings in sham treated mice suggest that
sgp130-Fc merits testing in models of mild and repetitive
mild TBI [55].
We did not observe a significant dose response with
sgp130-Fc treatment, which was the basis for combining the mice in those two dosing groups. Only a trend
toward slightly improved MWM metrics were observedwith our high versus lower dose regimen. An examination in future studies of multilevel dosing for otherbehavioral, histological as well as brain and spleenmolecular endpoints (e.g. RNA sequence techology) is
required to ensure an optimal dose titration is obtained
and its impacts on region and cell specific heterogeneitywith a complex injury model such as CCI [58–61]. Further work elucidating brain region specific influences ofclassical signaling (e.g. microglial) and trans-signaling(e.g. neuronal) on damage burden, plasticity, and repair
mechanisms is also warranted. Additional studies are alsoneeded to determine the pharmacokinetics and dynamics

Page 9 of 12

of both systemic and CNS sgp130-Fc as well as additionalsafety testing for long-term treatment and use.
While we identified beneficial effects of trans-signaling
blockade via systemic sgp130-FC administration over the
initial two weeks after CCI, it is possible that IL-6 transsignaling may play a neuroprotective role under certain
conditions post-injury. For example, Willis et al. showedthat in the context of microglial repopulation, IL-6 transsignaling may support neurogenesis and improve behavioral function in a mouse CCI model [62]. While theresults are intriguing, this model required full microglial turnover to obtain this effect along with a high-dose(2ug) intrahippocampal sgp130-Fc injection [62]. However, our work is consistent with other work demonstrating the detrimental effects of sIL-6R trans-signaling in
multiple neurodegenerative disease states, and the beneficial impacts of central sgp130 administration on sickness behaviors and associated cognitive dysfunction [7,15].

Conclusion
We demonstrated beneficial effects of systemic sgp130FC administration on behavioral performance and neuroinflammation after severe CCI in mice, with no adverse
impacts on mortality. The selective nature of sgp130-Fc
targets trans-signaling, leaving classical signaling intact.
While this exploratory report suggests possible therapeutic benefit, dose optimization in males and females,
further characterization of target engagement and additional neurorecovery outcomes require further examination. Overall, our data suggest that sgp130-Fc may be
a promising therapy for post-acute TBI recovery withpotential for future clinical applications.
Abbreviations
CCI	Controlled Cortical Impact
CNS	Central Nervous System
CSF	Cerebrospinal Fluid
D	Days
Fc	Fusion Protein
FI	Fluorescent Intensity
gp	Glycoprotein
HPA	Hypothalamic Pituitary Adrenal
IBD	Inflammatory Bowel Disease
ICAM	Intercellular Adhesion MoleculeIL	Interleukin
IP	Interferon γ-induced Protein
JAK/STAT	Janus Kinase/Signal Transducer and Activator of Transcriptionm	Membrane-bound
MCP	Monocyte Chemoattractant Protein
MHC	Major Histocompatibility Complex
MIG	Monokine Induced by Gammamin	Minutes
MIP	Macrophage Inflammatory Protein
ML	Mediolateral
MS	Multiple Sclerosis
MWM	Morris Water Maze
LPS	Lipopolysaccharide
PBS	Phosphate-Buffered Saline
R	Receptor
RA	Rheumatoid Arthritis

Gober et al. Journal of Neuroinflammation(2024) 21:149

s	Solublesec	Seconds
SEM	Standard Error of the Mean
TBI	Traumatic Brain Injury
TM	Trimmed Mean
VEH	Vehicle
VP	Visible Platform

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03129-0.
Supplemental Fig. 1. Effect of sgp130-Fc post-CCI on non-speed-adjusted
MWM metrics (a) Latencies to hidden platform (b) Peripheral zone time (c)Path length and during learning acquisition (D14-18) and VP (D19). Acquisition data were analyzed via mixed modeling for main effect and Sidak
post-hoc testing. VP was analyzed using linear regression. Black arrow on
D13 represents final sgp130-Fc or VEH administration. Lines/bars represent
mean ± SEM. Significant comparisons (p < 0.05) include: *Sham + VEH vs.
CCI + VEH, %Sham + VEH vs. CCI + sgp130-Fc, ^Sham + VEH vs. Sham + 1 µg
sgp130-Fc, #CCI + VEH vs. CCI + sgp130-Fc, +Sham + 1 µg sgp130-Fc vs.
CCI + VEH, !Sham + 1 µg sgp130-Fc vs. CCI + sgp130-Fc.
Supplemental Fig. 2. Effect of two doses of sgp130-Fc treatment after CCI
on MWM metrics. (a) Mean speed (b) speed adjusted escape latencies(c) speed adjusted peripheral zone time (d) speed adjusted path length
Learning acquisition data were analyzed via mixed modeling for main
effect and Sidak post-hoc testing. VP was analyzed using linear regression.
Black arrow on D13 represents final sgp130-Fc or VEH administration. Linesand bars represent mean ± SEM. Significant comparisons (p < 0.05) are as
follows: *Sham + VEH vs. CCI + VEH, %Sham + VEH vs. CCI + 0.25 µg sgp130Fc, $Sham + VEH vs. CCI + 1 µg sgp130-Fc, ^Sham + VEH vs. Sham + 1 µg
sgp130-Fc #CCI + VEH vs. CCI + 0.25 µg sgp130-Fc, @CCI + VEH vs. CCI + 1 µg
sgp130-Fc, &CCI + 0.25 µg sgp130-Fc vs. CCI + 1 µg sgp130-Fc, !Sham + 1 µg
sgp130-Fc vs. CCI + VEH, ?Sham + 1 µg sgp130-Fc + CCI + 0.25 µg sgp130-Fc,~
Sham + 1 µg sgp130-Fc + CCI + 1 µg sgp130-Fc.
Supplemental Fig. 3. Effect of two doses of sgp130-Fc after CCI on
non-speed adjusted MWM metrics. (a) Non-speed adjusted latencies (b) non-speed adjusted peripheral zone time and (c) non-speed
adjusted path length. Learning acquisition data were analyzed via mixed
modeling for main effect and Sidak post-hoc testings. VP was analyzed
using linear regression. Black arrow on D13 represents final sgp130-Fc
or VEH administration. Lines and bars represent mean ± SEM. Significant comparisons (p < 0.05) are as follows: *Sham + VEH vs. CCI + VEH,%
Sham + VEH vs. CCI + 0.25 µg sgp130-Fc, $Sham + VEH vs. CCI + 1 µg
sgp130-Fc, ^Sham + VEH vs. Sham + 1 µg sgp130-Fc #CCI + VEH vs.
CCI + 0.25 µg sgp130-Fc, @CCI + VEH vs. CCI + 1 µg sgp130-Fc, &CCI + 0.25 µg
sgp130-Fc vs. CCI + 1 µg sgp130-Fc, !Sham + 1 µg sgp130-Fc vs. CCI + VEH,?
Sham + 1 µg sgp130-Fc + CCI + 0.25 µg sgp130-Fc, ~Sham + 1 µg sgp130Fc + CCI + 1 µg sgp130-Fc.
Supplemental Fig. 4. Effect of two doses of sgp130-Fc after CCI on IL-6
related biomarkers and chemokines. (a) IL-6, (b) sIL-6R, (c) sgp130, (d) MIG,(e) IP-10, (f ) MIP-1β were analyzed with Kruskal-Wallis tests. Post-hoc comparisons used a Dunn’s Test. Significant comparisons (*) indicated p < 0.05.
Lines and bars represent mean ± SEM.
Acknowledgements
The authors would like to thank Denise Prosser (Luminex Core Laboratory
Manager at UPMC Hillman Cancer Center) for her assistance with the Luminex
assays and Joshua Michel (Senior Flow Core Technician, John G. Rangos
Research Center and Department of Pediatrics, University of Pittsburgh Schoolof Medicine) for his guidance on Luminex assay data analysis. We would also
like to thank Jessa Darwin (Research Project Manager, Department of Physical
Medicine and Rehabilitation, University of Pittsburgh School of Medicine)and Lara Nasser (Research Assistant, Department of Physical Medicine
and Rehabilitation, University of Pittsburgh School of Medicine School of
Medicine) for their assistance with the preparation of this manuscript.

Page 10 of 12

Author contributions
Conceptualization and study design: AKW; Investigation: IGG, TJS, VAV; Analysis:
IGG, JCC, AKW; Data interpretation: IGG, ALR, PMK, AKW; Writing - original draft:
IGG, ALR, AKW. Review and editing - original draft: JCC, PMK, AKW.
Funding
This work was funded by the University of Pittsburgh Medical Center
Rehabilitation Institute (AKW) and the University of Pittsburgh Physicians
Foundation (AKW).
Data availability
All data generated or analyzed during this study are included in this publishedarticle and its supplementary information files.

Declarations
Ethics approval and consent to participate
Animals were handled and treated in accordance with the National Institutesof Health Guide for the Care and Use of Laboratory Animals and wereapproved by the Institutional Animal Care and Use Committee (University ofPittsburgh).
Conflict of interest
The authors have no conflicts of interest to disclose.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Physical Medicine and Rehabilitation, School of Medicine,
University of Pittsburgh, 3471 Fifth Avenue, Suite 910, Pittsburgh,
PA 15213, USA
2
Safar Center for Resuscitation Research, John G. Rangos Research Center,
Pittsburgh, PA, USA
3
Department of Critical Care Medicine, School of Medicine, University ofPittsburgh, Pittsburgh, PA, USA
4
Center for Neuroscience, School of Medicine, University of Pittsburgh,
Pittsburgh, PA, USA
5
Department of Biostatistics, School of Public Health, University ofPittsburgh, Pittsburgh, PA, USA
6
Department of Human Genetics, School of Public Health, University ofPittsburgh, Pittsburgh, PA, USA
7
Department of Neuroscience, School of Arts and Sciences, University ofPittsburgh, Pittsburgh, PA, USA
8
Clinical and Translational Science Institute, University of Pittsburgh,
Pittsburgh, PA, USA
Received: 28 February 2024 / Accepted: 12 May 2024

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