Based on our prior research identifying macrophage infiltration in bladder biopsies from patients with benign prostatic obstruction through macrophage-specific gene expression markers, we hypothesize:
•	Pro-inflammatory M1 type macrophages play a significant role in the failing bladder as it transitions to advanced fibrosis, resulting in decreased contractility. 
We aimed to determine whether the deterioration of bladder function in MS patients is a mere consequence of disrupted neuronal pathways controlling micturition ("innocent bystander" scenario) or a reaction to the invasion of inflammatory immune cells, releasing cytokines which contribute to the emergence of overactivity, loss of contractility, underactivity and other symptoms (the “active participant” scenario)?

Our research aims to explore the involvement of pro-inflammatory macrophages in bladder dysfunction associated with both Multiple Sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). We conducted a proteomics analysis using mass spectrometry to identify pathways linked to macrophage function in EAE-afflicted bladders. Additionally, we investigated the balance between pro-inflammatory CCR2+ and anti-inflammatory CCR2- macrophages and their impact on the progression of lower urinary tract symptoms (LUTS) in MS. Our methods included flow cytometry, immunocytochemistry, and mRNA analysis to examine the presence and function of macrophage populations in bladder dysfunction. Specifically, we distinguished between resident and invading macrophages in EAE-induced mice both in vivo and ex vivo, utilizing CCR2-RFP x CX3CR1-GFP mice to elucidate their distinct roles in bladder dysfunction. By employing repeated urodynamic investigation (UDI) in awake mice, a technique established in our lab, we characterized bladder dysfunction in the EAE model of MS. We further studied the molecular composition of control and EAE mouse bladders using shotgun proteomics.

In EAE, bladder dysfunction manifests through increased bladder-to-body weight ratio and UDI recordings, correlating with symptomatic and morphological alterations. EAE mouse bladders exhibit heightened expression of pan-macrophage markers F4/80 and CD68, alongside elevated CCR2, indicating infiltration of pro-inflammatory M1 macrophages.
Proteomics analysis identifies activation of IL-12 signaling and production in macrophages, evidenced by changes in proteins such as APOM, COL1A1, COL1A2, COL2A1, COL3A1, GATA3, and ITGAM. Additionally, observed production of nitric oxide and reactive oxygen species in macrophages is influenced by alterations in APOM, ARG2, CYBB, MPO, NGFR, and RAC2 proteins. These findings suggest a significant role of macrophages in MS-associated LUTS development, particularly through fibrosis and hypoxia mechanisms. Proteomic discrepancies in EAE mice versus controls indicate organ remodeling, corroborated by pathway analysis of differentially expressed proteins (DEPs).
Furthermore, in EAE bladders, pathways related to extracellular matrix remodeling and fibrosis are notably activated, encompassing integrin cell surface interactions, collagen fibril assembly, collagen chain trimerization, extracellular matrix organization, hepatic fibrosis/hepatic stellate cell activation, collagen biosynthesis and modification, elastic fiber formation, and collagen degradation.

The findings from our study shed light on the intricate involvement of infiltrating macrophages in EAE-induced bladder dysfunction and its implications for Multiple Sclerosis (MS). Our results strongly support the hypothesis that pro-inflammatory M1 macrophages play a pivotal role in the progression of bladder dysfunction towards advanced fibrosis, leading to decreased contractility. This suggests that bladder dysfunction in MS patients may not solely be a consequence of disrupted neuronal pathways controlling micturition but could also be actively driven by the invasion of inflammatory immune cells, releasing cytokines that contribute to the emergence of symptoms such as overactivity, loss of contractility, and underactivity.
Furthermore, the activation of IL-12 signaling and production in macrophages, along with the observed alterations in proteins related to nitric oxide and reactive oxygen species production, indicates the involvement of macrophages in MS-associated lower urinary tract symptoms (LUTS), particularly through mechanisms involving fibrosis and hypoxia. The proteomic differences observed between EAE mice and controls further support the notion of organ remodeling in bladder dysfunction associated with MS. Pathway analysis of differentially expressed proteins highlights the activation of pathways related to extracellular matrix remodeling and fibrosis, emphasizing the complex interplay between immune cell infiltration and tissue remodeling processes.

Our study underscores the critical role of infiltrating macrophages in driving bladder dysfunction in the context of MS and EAE. Understanding the mechanisms underlying macrophage-mediated bladder pathology may pave the way for the development of targeted therapeutic interventions aimed at alleviating symptoms and improving quality of life for MS patients experiencing urinary dysfunction.

Continence 12S (2024) 101436
DOI: 10.1016/j.cont.2024.101436