Polymorphonuclear Cell Functional Impairment in Relapsing Remitting Multiple Sclerosis Patients: Preliminary Data

<div><p>Multiple Sclerosis patients run an increased risk of microbial infections, which leads to high rates of hospitalization and infection-related mortality. Although immunotherapy may increase infection risk in some cases, data as to the relationship among microbial factors, immunotherapy and alterations in the innate immunity of these patients are still scanty. On these grounds, this interdisciplinary study aims at investigating the role the functional activity of polymorphonuclear cells (PMNs) play in relapsing remitting multiple sclerosis at different stages. The <i>in vitro</i> ability of PMNs from patients, either untreated or treated with immunosuppressant or immunomodulatory drugs to kill <i>Klebsiella pneumonia</i> or <i>Candida albicans</i>, were investigated and compared to PMNs from healthy subjects. The release of various cytokines was also assessed, as was the production of reactive oxygen species and their ability to regulate apoptosis after microbial stimulation. Our results indicate that although patients have a normal number of PMNs, they have a statistically significant (p<0.05) reduction in intracellular killing activity. Although variations are strongly related to the therapeutic management of patients, they are independent from their disease stage. As no statistically significant differences were observed between patients and controls in cytokine release values, reactive oxygen species production or apoptosis, we came to the conclusion that other factors may be involved. Supportive validation of these results from further studies might well help in identifying a subset of patients at high risk of infection who could benefit from a closer follow-up and/or antibiotic prophylaxis.</p></div

Proinflammatory cytokine release by PMNs.

<p>(<b>A</b>) PMNs from 16 healthy subjects (HS), from 4 untreated-MS patients, from 6 patients treated with immunosuppressive drugs (IS-MS) or from 13 patients treated with immunomodulatory drugs (IM-MS) were stimulated with <i>K</i>. <i>pneumoniae</i> for 30, 60, 90 and 180 minutes. (<b>B</b>) PMNs from 10 healthy subjects (HS), from 7 untreated-MS patients, from 7 patients treated with immunosuppressive drugs (IS-MS) or from 9 patients treated with immunomodulatory drugs (IM-MS) were stimulated with <i>C</i>. <i>albicans</i> for 30, 60, 90 and 180 minutes. Supernatants were evaluated for IL-8 (upper panels) and IL-1β release (lower panels). Data are represented as average ± SEM. *p<0.05.</p

The analysis of induced oxidative burst in MS patients.

<p>Whole blood from healthy subjects (HS; n = 12), from untreated-MS patients (n = 7), from patients treated with immunosuppressive drugs (IS-MS; n = 6) or with immunomodulatory drugs (IM-MS; n = 12), was stimulated with <i>K</i>. <i>pneumoniae</i> (<b>A</b>), <i>C</i>. <i>albicans</i> (<b>B</b>) or PMA (<b>C</b>) for 30 minutes. Percentage of positive cells for DHR123 (left panels) or average of fluorescence intensity (MFI) of DHR123 (right panels) were determined by flow cytometry. Data are represented as average ± SEM.</p

The study of PMN apoptosis in MS patients.

<p>(<b>A</b>) The percentage of apoptotic purified PMNs (annexin V positive, PI negative) in healthy subjects (HS; n = 8), in untreated-MS patients (n = 6), in patients treated with immunosuppressive drugs (IS-MS; n = 5) or with immunomodulatory drugs (IM-MS; n = 10), after 3 hours of culture. (<b>B</b>) Early apoptotic cells were evaluated in HS (n = 5) and IM-MS (n = 5) PMNs after <i>K</i>. <i>pneumoniae</i> stimulation. Values were determined by flow cytometric analysis and data represented as average ± SEM. *p<0.05; **p<0.001</p

Demographic and clinical information on the healthy subjects (HSs) and MS patients.

<p>Average ± SEM are shown; EDSS: Kurtzke Expanded Disability Status Scale; RR: relapsing-remitting MS; UTIs: urinary tract infections; RTIs: respiratory tract infections</p><p>Demographic and clinical information on the healthy subjects (HSs) and MS patients.</p

Induction of selected cytokines and chemokines by OM-85 in MoDC.

<p><b>A</b>) MoDC (10⁶/ml) were stimulated with OM-85 as indicated and with 100 ng/ml LPS (IL-6, BAFF, CCL2, CXCL8 and CXCL6) or 10 ng/ml LPS (CCL3, CCL20 and CCL22) as a positive control. After 24 hours, supernatants were collected and analyzed by ELISA. Ut = untreated. *P<0.05 and **P<0.01 by Dunnett's Multiple Comparison Test. <b>B</b>) Supernatants of MoDC stimulated with OM-85 induce a G-protein-dependent migration of PMN. As a comparison, migration of PMN was elicited with unstimulated supernatants+CXCL8 and PMA. As expected, migration to CXCL8 was inhibited by both 10 nM M3 and 750 ng/ml, <i>Pertussis toxin</i> (P.Tox) while migration to PMA was not. Results are expressed as chemotactic index over migration to supernatants of unstimulated MoDC and represent means+/−SD of three independent Boyden chamber experiments. *P value<0.05 and ** P value<0.01 by Dunnett's Multiple Comparison Test.</p

Activation of PBMC and MoDC from COPD patients and healthy subjects by OM-85.

<p><b>A</b>) PBMC and <b>B</b>) MoDC (both 10⁶/ml) were stimulated as indicated in the presence or absence of 500 U/ml IFNγ or 100 ng/ml TNF-α. After 24 hours, supernatants were collected and analyzed by ELISA. Figure shows the results of healthy donors (open histograms) compared to COPD patients (black histograms). *P<0.05 by paired Student's <i>t</i> test.</p

Synergism between OM-85 and pro-inflammatory agonists in MoDC.

<p>MoDC (10⁶/ml) were stimulated as indicated. After 24 hours, supernatants were collected and analyzed by ELISA. *P<0.05 and **P<0.005 by paired Student's <i>t</i> test.</p

Activation of the NF-kB and MAPK pathways by OM-85 in MoDC.

<p><b>A</b>) Immature human MoDC were stimulated with 100 µg/ml OM-85 for 30, 60, 90 and 120 minutes. 100 ng/ml LPS was used as a positive control. After cell lysis and protein fractionation, cytoplasmic (Cyto) and nuclear (Nuclei) extracts were blotted against NF-kB p65 and IkBα. β-actin and Lamin A/C represent loading controls for cytoplasmic and nuclear proteins respectively. The image depicts results obtained in one representative donor out of eight. <b>B</b>) EMSA (upper panel) and supershift (lower panel) showing the induction of NFkBp65-DNA binding activity by OM-85 in human moDC stimulated as in A). Signal specificity was assessed by competing each sample with a 125-fold excess unlabeled probe (lanes 2,4,6,8,10 upper panel). The image depicts results obtained in one representative donor out of four. <b>C</b>) OM-85 induces the production of luciferase in THP1 cells bearing a NF-kB-reporter plasmid (NF-kB pGL4, striped histograms). THP1 cells were stimulated with 1 µg/ml LPS and 1000 µg/ml OM-85. As expected, THP1 untransfected cells (untransfected, empty histograms) did not produce luciferase in response to stimulation. Similar results were obtained when cells were transfected with the pGL4 empty backbone (pGL4, black histograms). Results are expressed as mean+/−SD of three independent experiments. *P value<0.01 by Dunnett's Multiple Comparison Test. <b>D</b>) OM-85 activates the MAPK pathway. Cell extracts prepared as in A) were blotted with antibodies specific for phophorilated ERK1/2 (Cyto, upper panel) and total ATF2 and c-Jun (Nuclei, lower panel). β-actin and Lamin A/C represent loading controls for cytoplasmic and nuclear proteins respectively. The image depicts results obtained in one representative donor out of three. <b>E</b>) OM-85 induces NF-kB- and MAPK-dependent gene transcription. Immature MoDC were stimulated with 100 µg/ml OM-85 (open circles) and 100 ng/ml LPS (black circles) for 2, 4, 8 and 24 hours. After RNA extraction, reverse transcription and DNAse I digestion, samples were amplified by Q-PCR using gene-specific primers. Results represent means+/−SE of three independent donors and are expressed as fold induction (FI) over unstimulated samples (0).</p

Activation of primary DC subsets by OM-85.

<p><b>A</b>) MDC and <b>B</b>) PDC were isolated from buffy coats of three independent healthy donors and stimulated as indicated (10⁶/ml). After 24 hours, supernatants were collected and analyzed by ELISA. *P<0.05 by Dunnett's Multiple Comparison Test.</p