Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: “SMALL FIBER NEUROPATHY EVALUATION IN SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS: A CLINICAL, NEUROPHYSIOLOGICAL AND HISTOPATHOLOGICAL STUDY”

Introduction: Neuropsychiatric Systemic Lupus Erythematosus (NPSLE) is considered one of the preeminent diagnostic and therapeutic challenges for the rheumatologist, due to the lack of specific diagnostic tools and biomarkers and its wide clinical heterogeneity. Recently American College of Rheumatology (ACR) 1999 NPSLE classification and case definition has been questioned, suggesting the inclusion of other manifestations, who came to attention more recently. Among them, small fiber neuropathy (SFN), characterized by the selective damage of the smallest sensory peripheral nerve, has been suggested as a main reason for chronic pain in SLE patients. SFN-related symptoms include neuropathic pain with paresthesia, allodynia, hyperalgesia and even insensitivity to pain. The pathogenic mechanisms of this manifestation are still largely unexplained in SLE, even though different autoantibodies and increased levels of proinflammatory cytokines have been associated with idiopathic SNF, suggesting the potential role of the immune system in its development. Aim: In our study we aimed at assessing SFN in SLE patients with painful disturbances and at identifying clinical, histopathological and laboratory features (including novel potential biomarkers) associated with this neurological manifestation. Patients and methods: From January 2020 to July 2022, 235 consecutive SLE patients (according to ACR 1987 criteria) with painful disturbances, were screened for neuropathic pain through a specific questionnaire (Douleur Neuropathique en 4 Questions-DN4) at our Lupus Clinic of Sapienza University of Rome, Policlinico Umberto I in Rome. Subjects were enrolled to the full protocol in the presence of selectively distal or widespread distribution and one of the following features: hypoesthesia to touch or prick; pain caused or increased by brushing; tingling, numbness and itching; electric shocks, painful cold or burning pain. We excluded SLE patients younger than 18 years, with severe cognitive disturbances, and psychiatric disorders as assessed through clinical history and examination. Moreover we ruled out, through complete clinical and laboratory evaluation, every other possible explanation for SFN including diabetes, other autoimmune disease (in particular Sjogren’s syndrome or celiac disease), kidney impairment (eGFR <30 ml/m), vitamin B12 deficiency, hypothyroidism, previous neurotoxic treatment and alcohol abuse. For each patient we collected the main clinical, demographic and laboratory data. In collaboration with the Neuroscience Department, each enrolled patient underwent a nerve conduction study (NCS), quantitative sensory testing (QST) and skin punch biopsy, performed on two different sites (leg and thigh). Tissue specimen was devoted to the following evaluations. Firstly it was dedicated to immunofluorescence staining with PGP 9.5 to study intra-epidermal nerve fibers density and C3 to evaluate complement deposition; later on it was devoted to immunohistochemical staining with CD45 leukocyte common antigen and proteomic analysis in order to identify potential new autoantigens specific for SFN. The diagnosis of SFN rely on normal NCS and was made according to the Besta criteria, meeting at least two abnormal findings among the three following: the presence of not less than two clinical signs of small fiber impairment (pinprick and thermal sensory loss and/or allodynia and/or hyperalgesia); abnormal warm and/or cold detection thresholds at QST; reduced Intraepidermal Nerve Fiber Density (IENFD) at skin biopsy. In order to evaluate the impact of fibromyalgia on chronic pain in SLE patients, its diagnosis was based on the ACR 2016 criteria. All patients underwent specific questionnaires on small fiber related symptoms and fibromyalgia symptoms. Results: In the end, 50 subjects have been enrolled (F/M 46/4; median age 50, IQR 18.75; median disease duration 132 months, IQR 177). Out of 50 SLE patients, 52% showed diagnostic findings compatible with neuropathy: 38% had normal NCS and fulfilled the Besta criteria, attributable to SFN, while 14% had NCS abnormalities and fulfilled Besta criteria, attributable to a mixed fiber neuropathy (MFN), with both large and small fiber involvement. Out of 19 patients with SFN, 89% showed non-length dependent ganglionopathy-like fibers reduction pattern. MNF patients were older than other subjects and patients with isolated SFN (p=0.002 and 0.0072 respectively). Moreover, disease duration negatively correlated with large fibers mediated variables, like sural nerve sensory action potential amplitude (p=0.0018; r=-0.448). As expected, NCS abnormalities and also MFN were significantly more common in patients with previous NPSLE manifestations (p=0.0030 and p=0.0028). Patients with SFN more frequently presented hypocomplementemia in their history than patients without neuropathy (p= 0.045 and p=0.003, when considering only isolated SFN). Moreover low complement levels were associated with low IENFD at the proximal site (p=0.009). SFN patients had more commonly been treated with cyclosporine in their clinical history (p=0.039 and p=0.003 when considered only isolated SFN). Lower IENFD level was found at distal sites in patients with chronic damage than in patients with SDI values equal to 0 (p=0.023). A significant association was found between IENFD at both distal and proximal sites with neuropathic symptoms reported though SFN-SIQ (p=0.006, r-0.40, p=0.010 r=-0.37, respectively). Patients with SFN showed a significant reduction of Piloerector Muscle Nerve Fiber Density (PMNFD) and Sweat Gland Nerve Fiber Density (SGNFD) both at distal and proximal sites than healthy controls (distal PMNFD: p<0.0044; proximal PMNFD: p=0.0364; distal SGNFD: p<0.0001; proximal SGNFD: p=0.0052). Distal PMNFD negatively correlated with Raynaud phenomenon (p=0.0400; r=-0.353). Conclusion: Our study confirms that SFN with a non-length dependent distribution is a highly prevalent manifestation in SLE patients with chronic pain, much more common than large fiber neuropathy; therefore it should be considered as a possible NPSLE manifestation even if not considered in ACR case definitions yet. In our opinion small fiber testing should be implemented in the assessment and follow up of SLE patients with neuropathic pain, since SFN likely precedes the development of a MFN and is very difficult to identify if not specifically investigated. The association with hypocomplementemia and chronic damage strengthen the hypothesis of a causal link between SLE and SFN, and suggest the role of immune system dysfunction in the development of this neuropathy. Moreover, the association with Raynaud phenomenoncould suggest that SFN may underlie multiple clinical manifestations related to SLE.