Anti-Pituitary and Anti-Hypothalamus Autoantibody Associations with Inflammation and Persistent Hypogonadotropic Hypogonadism in Men with Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) and can lead to persistent hypogonadotropic hypogonadism (PHH) and poor outcomes. We hypothesized that autoimmune and inflammatory mechanisms contribute to PHH pathogenesis. Men with moderate-to-severe TBI (n = 143) were compared with healthy men (n = 39). The TBI group provided blood samples 1-12 months post-injury (n = 1225). TBI and healthy control (n = 39) samples were assayed for testosterone (T) and luteinizing hormone (LH) to adjudicate PHH status. TBI samples 1-6 months post-injury and control samples were assayed for immunoglobulin M (IgM)/immunoglobulin G (IgG) anti-pituitary autoantibodies (APA) and anti-hypothalamus autoantibodies (AHA). Tissue antigen specificity for APA and AHA was confirmed via immunohistochemistry (IHC). IgM and IgG autoantibodies for glial fibrillary acid protein (GFAP) (AGA) were evaluated to gauge APA and AHA production as a generalized autoimmune response to TBI and to evaluate the specificity of APA and AHA to PHH status. An inflammatory marker panel was used to assess relationships to autoantibody profiles and PHH status. Fifty-one men with TBI (36%) had PHH. An age-related decline in T levels by both TBI and PHH status were observed. Injured men had higher APA IgM, APA IgG, AHA IgM, AHA IgG, AGA IgM, and AGA IgG than controls (p < 0.0001 all comparisons). However, only APA IgM (p = 0.03) and AHA IgM (p = 0.03) levels were lower in the PHH than in the non-PHH group in multivariate analysis. There were no differences in IgG levels by PHH status. Multiple inflammatory markers were positively correlated with IgM autoantibody production. PHH was associated with higher soluble tumor-necrosis-factor receptors I/II, (sTNFRI, sTNFRII), regulated on activation, normal T-cell expressed and secreted (RANTES) and soluble interleukin-2-receptor-alpha (sIL-2Rα) levels. Higher IgM APA, and AHA, but not AGA, in the absence of PHH may suggest a beneficial or reparative role for neuroendocrine tissue-specific IgM autoantibody production against PHH development post-TBI.

Keywords: IgG autoantibody; IgM autoantibody; TBI; autoantibodies; autoimmunity; hypogonadism; hypopituitarism; inflammation.

FIG. 1.

Consolidated Standards of Reporting Trials (CONSORT) flow chart. Out of the 143 men adjudicated for persistent hypogonadotropic hypogonadism (PHH), 137 had autoantibody data and 138 had inflammation data, and 132 participants had both autoantibody and inflammation data. Out of the 137 with autoantibody data, 124 individuals received trajectory group assignments. AAb, autoantibody; TRAJ, group-based trajectory analysis.

FIG. 2.

Immunoglobulin (Ig)M and IgG autoantibody standard curves. IgM and IgG class autoantibodies enzyme-linked immunosorbent assays (ELISA) standard curves used to calculate the anti-pituitary autoantibodies (APA), anti-hypothalamus autoantibodies (AHA), and anti- glial fibrillary acid protein (GFAP) autoantibody concentrations (titers) in human subject serum. (A) IgM standard curves, (B) IgG standard curves. Calculated IgG or IgM concentrations ± standard deviation (STD) from four independent runs were shown. Linear regression fitting results are shown.

FIG. 3.

Frequency distribution for (A) duration of psychotropic medication use and (B) Cumulative psychotropic medication burden (aggregate measure of medication use and duration of each medication) during the first year post-injury for individuals categorized by persistent hypogonadotropic hypogonadism (PHH) status.

FIG. 4.

Scatter plot of serum testosterone (T) and luteinizing hormone (LH) by age in the traumatic brain injury (TBI) cohort. Linear regression for (A) 1–6 month average T levels. Also graphed are mean T levels for our healthy controls (16.64 nmol/L) and our clinical laboratory low-normal reference level (10 nmol/L). (B) 1–6 month average LH levels compared with age for men with TBI. There was a significant reduction in serum T with older age (p = 0.0001) but only a trend for increases in LH with older age (p = 0.0521). Also graphed are mean LH (4.82 IU/L) for healthy controls and high normal reference level for our clinical laboratory (5.6 IU/L).

FIG. 5.

Mean traumatic brain injury (TBI) (n = 143) and control (n = 11) and testosterone (T) and luteinizing hormone (LH) levels stratified by persistent hypogonadotropic hypogonadism (PHH) status. Individuals in the TBI group were stratified by PHH status. (A)T levels are higher for controls versus all TBI and versus the TBI PHH group. Those without PHH (n = 92) had higher T versus those with PHH (n = 51). (B) Serum LH levels averaged 1–6 months post-injury for TBI (n = 143) versus controls (n = 10). Controls and non-PHH TBI groups (n = 92) had higher mean LH levels than men with PHH (n = 51). #Our clinical laboratory low-normal T reference level (10 nmol/L) is also graphed as is our clinical laboratory high normal for LH (5.6IU/L). (A, B) #p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as bar graphs + standard error of mean.

FIG. 6.

Mean control (n = 39) and traumatic brain injury (TBI) (n = 137) anti-pituitary antibody (APA) and anti-hypothalamic antibody (AHA) immunoglobulin (Ig)M and IgG levels stratified by persistent hypogonadotropic hypogonadism (PHH) Status. APA IgM concentrations tended to be lower in the PHH group than in the non-PHH group (A). There is also significantly lower AHA IgM expression in PHH than in non-PHH (B). No statistically significant differences existed between PHH and non-PHH groups in APA IgG, or AHA IgG (C and D). There were significant elevations in mean 1–6-month Ig concentrations in both IgM and IgG APA and AHA with TBI versus controls. The non-PHH group had higher APA and AHA IgM and IgG levels than controls. (A–D) ^#p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as bar graphs + standard error of mean.

FIG. 7.

Mean immunoglobulin (Ig)M and IgG autoantibody levels for glial fibrillary acid protein (GFAP) for traumatic brain injury (TBI) (n = 137) and control (n = 39) groups as well as for TBI group stratified by persistent hypogonadotropic hypogonadism (PHH) status. (A) Anti-GFAP autoantibodies are increased for TBI group versus control, but levels did not differ by PHH status. (B) IgG GFAP autoantibodies are increased for TBI group versus control, but levels did not differ by PHH status. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as bar graphs + standard error of mean.

FIG. 8.

Immunoglobulin (Ig) levels by group-based trajectory analysis (TRAJ) group membership. (A) IgM anti-pituitary antibody (APA) and (B) anti-hypothalamic antibody (AHA) levels were graphed by the generated TRAJ groups. There were significant differences in autoantibody levels by TRAJ groups for all autoantibodies (A and B, p < 0.05 all comparisons). Dashed lines represent mean control levels (APA IgM, 0.24 μg/mL and AHA IgM, 4.32 μg/mL).

FIG. 9.

Mean testosterone (T) Levels by immunoglobulin (Ig)M anti-pituitary antibody (APA) (A) and IgM anti-hypothalamic antibody (AHA) (B) group-based trajectory analysis (TRAJ) group membership. T levels were graphed by IgM TRAJ group membership. T levels are significantly different over time and T levels differ by high versus low IgM TRAJ groups (A and B). T levels follow the same overall pattern longitudinally across both high and low APA/AHA IgM TRAJ groups. Dashed lines represent mean control levels (T = 16.64 nmol/L). The clinical laboratory low-normal reference level (10 nmol/L) is also graphed.

FIG. 10.

Anti-pituitary antibody (APA) and anti-hypothalamic antibody (AHA) immunoglobulin (Ig)M fluorescence immunohistochemistry (IHC) staining with selected traumatic brain injury (TBI) participants. Human cavaderic pituitary and hypothalamic tissue was stained with subacute-chronic (1 month) serum samples from four men (subjects 1–4) with moderate to severe TBI. Each serum sample was exposed to human pituitary and hypothalamic tissue sections and then developed with fluorophore Alexa 555-conjugated anti-human IgM (images shown from top to bottom in each column). Yellow arrows point to strongly staining cells. White bar in lower right corner represents 50 μm. Individual subject's membership in APA and AHA IgM TRAJ group (high, low) are shown on top and bottom, respectively, of its IHC images.

FIG. 11.

Anti-hypothalamic antibody (AHA) immunoglobulin (Ig)M mediation model. This brain injury severity adjusted model demonstrates a mediation effect (by 31.91%) on the relationship between age and persistent hypogonadotropic hypogonadism (PHH) by AHA IgM levels. Increasing age was associated with lower levels of AHA IgM (month 1–6 means) and PHH. Greater month 1–6 mean AHA IgM levels were also associated with reduced odds of PHH. Each model leg adjusted for best in 24 h. Glasgow Coma Scale (GCS) score. Statistical significance denoted by: *p < 0.05, **p < 0.01. CI: confidence interval OR: odds ratio; SE: standard error.

FIG. 12.

Cytokine biomarkers associated with persistent hypogonadotropic hypogonadism (PHH) status. Of the 33 inflammatory markers assayed, the five graphed above were significantly associated with PHH status (p < 0.05). Each marker was scaled by a multiple of 10^x (indicated in the graph) to fit a 0–20 pg/mL range. Only granulocyte macrophage colony stimulating factor (GM-CSF) levels were higher in non-PHH individuals, while soluble tumor-necrosis-factor receptors (sTNFR)I, sTNFRII, regulated on activation, normal T-cell expresse (RANTES), and soluble interleukin receptor (sIL-2R)α levels were significantly higher in PHH.