Neuroinflammation and osteomyelitis in adults with Type 2 diabetes mellitus and peripheral neuropathy without and with foot lesions. What comes first?

Aims: Diabetic foot is the leading cause of both major and minor non-traumatic amputations yet a truly understanding of the phenomenon is still lacking. The updated definition for diabetes-related foot disease from the International Working Group on the Diabetic Foot (IWGDF 2023 update) is “disease of the foot of a person with current or previously diagnosed diabetes mellitus that includes one or more of the following: peripheral neuropathy, peripheral artery disease, infection, ulcer(s), neuro‐osteoarthropathy, gangrene, or amputation”, but what comes first? Our hypothesis is that distal sensory and autonomic neuropathy activate neuroischemic signaling and dysregulation of bone cell apoptosis portending to infections. Methods: We studied 374 adults with Type 2 diabetes mellitus (T2DM) and diabetic neuropathy (DN) divided into the following subgroups: 106 partecipants without foot lesions (DNp); 119 nonmacrovascular partecipants with ulcers/lesions/osteomyelitis (DNpU); 149 revascularized partecipants with ulcers/lesions/osteomyelitis (DNpUV) and a group of 53 healthy adults as healthy control (NC). During routine foot care visits participants underwent neuro electrophysiology tests and vascular assessment. Biopsy specimens from exposed bone (grade III University of Texas wound classification, TUC) were cultured according to microbiological standards and histological analysis was performed. Pro/anti-inflammatory cytokines and blood cells subsets (lymphocytes subpopulations, classical, non-classical and SLAN+ monocytes, classical DCs, innate lymphoid cells) were analyzed. Nerve Growth Factor (NGF) species, fractalkine/CX3CL1 migration marker, autophagy markers (Ulk1, Beclin1, LC3, and p62), pro/anti-apoptotic proteins (Bax, Bcl2, cleaved Caspase-3), signal transduction of proteins involved in inflammation and cell survival (p65-NF-kB, Akt and ERK1/2) were measured. Results: Sural nerve (sS) conduction velocity (CV) and sensory Action Potential (sAP) thresholds defined DN. II and III TUC associates with progressive worsening of neuronal function while vibration perception threshold (VPT) and systolic/diastolic orthostatic hypotension inversely correlated with TcPO2 and critical ischemia. In III TUC versus I TUC diabetic foot ulcer (DFU) and DNp samples the amount of circulating mature NGF (mNGF) was significantly reduced (p < 0.01) while immature NGF (proNGF) was significantly increased (p < 0.05). In all groups we found higher number of SLAN+ monocytes co-expressing CX3CR1 directly correlating with proNGF levels, worse autonomic and sensory testing and inversely correlating with mNGF levels, sensory nerves CV and AP, innate lymphoid cells and subsets of lymphocytes. Surprisingly, we found 59 bone's biopsies with an altered histological pattern but negative microbiological cultures. In all biopsied patients CX3CR1-SLAN+ cells were significantly elevated (p < 0.05) independently of concomitant infective osteomyelitis in III TUC versus the level measured in I TUC and DNp. Cleaved caspase 3 and Bax directly correlated with % of CX3CR1+SLAN+CD16+ inflammatory monocytes and inversely with bone ERK1/2 activating phosphorylation. Conclusions: DN could be an important factor in diabetic foot. Reduced proprioception and sympathetic orthostatic hypotension destroy structures of the foot, alter intrabone neurotrophins resulting in vascular signaling, early activation of bone apoptosis and CX3CR1+monocytes prior to and independently of infected osteomyelitis. These data suggest a new interpretation of the main players involved in the physiopathology of diabetic foot and may mark the way for the development of new targeted therapies.