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. 2024 Apr 19;25(8):4484.
doi: 10.3390/ijms25084484.

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study

Shaojing Ye  1 , Nilesh M Agalave  2 , Fei Ma  1 , Dlovan F D Mahmood  1 , Asma Al-Grety  2 , Payam E Khoonsari  2 , Lin Leng  3 , Camilla I Svensson  4 , Richard Bucala  3 , Kim Kultima  2 , Pedro L Vera  1   5
Affiliations

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study

Shaojing Ye et al. Int J Mol Sci. .

Abstract

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 μg); or MIF mAb (15 μg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Keywords: CD74; CXCR4; macrophage migration inhibitory factor; persistent bladder pain; spinal proteins.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of intrathecal MIF antagonism on persistent bladder pain. The 50% threshold to von Frey stimulation is depicted on the y-axis, while the time in days is depicted on the x-axis. Insets (green arrows) show the time in hours post-intrathecal injection. Differences in threshold after treatment were determined using repeated measures ANOVA followed by Dunnett’s post hoc tests (compared to time 0). Repeated (3×; red arrows) intravesical instillation of PAR4-AP resulted in marked decrease in threshold (an index of bladder pain) that recovered on day 2 but remained decreased after subsequent instillations and persisted until day 9. This is our index for persistent bladder pain. The effects of intrathecal (L6/S1) injections on day 7 were assessed using repeated-measures ANOVA followed by post hoc Dunnett’s tests comparing all the time periods to 0. (A) Intrathecal injection of isotype control had no effect on decreased von Frey threshold, and consequently no effect on established persistent BHA. (B) Intrathecal injection of a neutralizing MIF mAb quickly reversed established persistent BHA (as shown by the increased threshold), with a maximal effect at 2 h post-injection. * = 0.05, ** = 0.01, *** = 0.001. The insets show the von Frey threshold for up to 6 h after i.t. injection.
Figure 2
Figure 2
Boxplot showing the median and interquartile range (the mean is displayed as the red diamond) and dotplot of the micturition volume and frequency for each group as evaluated on day 9 of the protocol. Differences between the two groups were evaluated using t-tests. (A) The micturition volume (in µL) shows no significant difference between the two groups (p = 0.40). (B) The micturition frequency (number of micturitions/3 h) is identical between the two groups; therefore, no significant difference was observed (p = 1).
Figure 3
Figure 3
Histological examination of bladder inflammation and edema. Bladders were collected on day 9 (end of experiment), processed for H&E staining, and scored for inflammation and edema as listed above. Representative sections are presented for each group. There is little or no evidence of urothelial damage and/or suburothelial inflammation and/or edema in mice from either group. (A) Mice treated with 15 µg IgG1 (isotype control) intrathecally. (B) Mice treated with 15 µg anti-MIF monoclonal antibody intrathecally. Calibration bar = 200 µm.
Figure 4
Figure 4
Boxplots showing median and interquartile range along with dotplots for inflammation and edema scores. Differences between the two groups were assessed using Fisher’s exact test. (A) There is no statistical difference (p = 0.52) in inflammation scores between mice treated with intrathecal IgG1 vs intrathecal MIF neutralizing antibody (MIF mAb). (B) Edema scores between the two groups do not show a statistically significant difference (p = 0.71).
Figure 5
Figure 5
Effect of intrathecal antagonism of MIF receptors on persistent bladder pain. The 50% threshold to von Frey stimulation is depicted on the y-axis, while the time in days is depicted on the x-axis. Red arrows indicate days of intravesical PAR4 installation. Insets (green arrows) show the time in hours post-intrathecal injection. Differences in threshold after treatment were determined using repeated measures ANOVA followed by Dunnett’s post hoc tests (compared to time 0). (A) Intrathecal treatment with anti-CD74 mAb had a small yet significant (* = p < 0.05) reversal of persistent BHA at 4 h post-injection. Intrathecal treatment with rat IgG2b (isotype control) showed a small significant decrease in threshold at 1 h (* = p < 0.05). (B) Intrathecal administration of AMD3100 (CXCR4 antagonist) on day 7 partially reversed persistent BHA, with the peak effect occurring 4 h after treatment (*** = p < 0.001). Intrathecal treatment with PBS (vehicle control) on day 9 had no effect on persistent BHA (p = 0.29). (C) The effects of the three spinal antagonists (MIF mAb, CD74 mAb, and AMD3100) were evaluated using ANOVA followed by Dunnett’s post hoc tests. Both CD74 mAb and AMD3100 showed significant differences (### = p < 0.001) when compared to the response to MIF mAb. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 when compared to 0 hours.
Figure 6
Figure 6
Protein levels were compared using ratios between samples, then pools and data were transformed to log2 scale (y-axis). ANOVA was used to determine whether any significant differences existed between the groups. Post hoc tests (t-test) were used to compare the estimated marginal means between all possible contrasts (pairs of groups). Because all ratios are normalized to control, the protein ratio for the control group is not shown. Each panel shows the estimated marginal means and standard error of the mean for each group along with the t-test significance compared to control. Novel MIF-modulated proteins were determined by selecting proteins in the persistent (sham i.t.; Group 2; red) and unalleviated BHA (isotype IgG1 i.t; blue, Group 3) groups that showed a significant difference when compared to control group (no pain; Group 1) and no significant difference when MIF mAb i.t. treatment was compared to control. (A) Persistent and unalleviated BHA (red; blue) showed decreased levels in six proteins depicted and increased levels in two proteins (B). These changes were reversed by intrathecal treatment with a MIF mAb (green; Group 4). All ratios are normalized to the control group.
Figure 7
Figure 7
Decreased V-set transmembrane domain-containing protein 2-like (VSTM2L) immunostaining in L6/S1 spinal cord in the persistent BHA group. (A) Representative image of VSTM2L immunostaining image from a dorsal horn of a mouse treated with scrambled peptide (no pain). Positive cells (presumably neurons) can be seen in the dorsal horn. (B) Representative image of VSTM2L immunostaining in the dorsal horn area from a mouse treated with repeated instillations of PAR4-AP to induce persistent BHA. A significant decrease in staining of the cells in the dorsal horn support the findings from the proteomics analysis. (C) For each spinal cord section, immunopositive neurons in both dorsal horns were counted and summed; a boxplot showing the median and interquartile range (with the mean displayed by the red diamond) along with a dotplot is presented for both treatment groups. There was a significant decrease (* p < 0.05) in the number of VSTM2L positive cells in the PAR4-AP (PAR4) group.
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