Intranasal Application of Lactococcus lactis W136 Is Safe in Chronic Rhinosinusitis Patients With Previous Sinus Surgery.

Objective: Modulation of the dysbiotic gut microbiome with "healthy" bacteria via a stool transplant or supplementation is increasingly practiced, however this approach has not been explored in the nasal passages. We wished to verify whether Lactococcus lactis W136 (L. lactis W136) bacteria could be safely applied via irrigation to the nasal and sinus passages in individuals with chronic rhinosinusitis (CRS) with previous undergone endoscopic sinus surgery, and whether this was accompanied by bacterial community flora modification. Study Design: Prospective open-label pilot trial of safety and feasibility. Setting: Academic tertiary hospital center. Subjects and Methods: Twenty-four patients with CRS refractory to previous medical and surgical therapy received a 14-day course of BID sinus irrigations containing 1.2 × 109 CFU live L. lactis W136. Patients were monitored for safety using questionnaire, sinus endoscopy, otoscopy, UPSIT-40 smell testing, and endoscopically-obtained conventional sinus culture and a swab for 16S microbiome profiling. Results: All 24 patients receiving at least one treatment successfully completed treatment. L. lactis W136 probiotic treatment was safe, with no major adverse events or new infections. Treatment was associated with improvement in sinus symptoms, QOL, and mucosal scores, which remained improved during the subsequent 14-day observation period. Microbiome changes associated with treatment were limited to an increase of the pathobiont Dolosigranulum pigrum, a bacteria identified as potentially beneficial in the upper airways. Subgroup analysis suggested differences in microbiomes and responses for CRSsNP and CRSwNP phenotypes, but these did not attain significance. Conclusion: Intranasal irrigation of live L. lactis W136 bacteria to patients with refractory chronic rhinosinusitis was safe, and was associated with effects on symptoms, mucosal aspect and microbiome composition. Intranasal bacteria may thus find a role as a treatment strategy for CRS. Clinical Trials Registration: www.ClinicalTrials.gov. identifier: NCT04048174.

Topical probiotics as a therapeutic alternative for chronic rhinosinusitis: A preclinical proof of concept.

INTRODUCTION: Patients with chronic rhinosinusitis (CRS) have been shown to manifest a high inflammatory phenotype, with a sinus microbiome deficient in gram-positive bacteria. Gram-positive bacteria are capable of downregulating proinflammatory host responses via an interleukin (IL) 10 mediated response and may represent a potential therapeutic alternative for CRS. We wanted to (i) immunoprofile the IL-10 induction capacity of two gram-positive probiotic strains and (ii) verify the tolerance of these strains by the sinus epithelium. METHODS: A peripheral blood mononuclear cell (PBMC) challenge model was used to document probiotic induction of IL-10 and tumor necrosis factor (TNF) alpha responses at various bacterial dilutions. Epithelial cell tolerance was demonstrated by using a primary epithelial cell model derived from patient biopsy specimens (six patients total [three with CRS and three controls]). After an incubation period with either a live or a heat-killed probiotic strain, cell viability was assessed by using light microscopy. RESULTS: Both probiotic strains induced high IL-10 secretion in PBMCs, with differing profiles of TNF alpha production. Microscopic evaluation after probiotic incubation demonstrated intact cell viability for all cell cultures. CONCLUSION: We identified well-tolerated, nonpathogenic, "generally recognized as safe" status gram-positive probiotics with anti-inflammatory properties. Topical probiotics represented a potential novel topical therapeutic strategy for CRS relevant for further clinical evaluation.

Uncoupling of pro- and anti-inflammatory properties of Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that is carried by a quarter of the healthy human population and that can cause severe infections. This pathobiosis has been linked to a balance between Toll-like receptor 2 (TLR2)-dependent pro- and anti-inflammatory responses. The relationship between these two types of responses is unknown. Analysis of 16 nasal isolates of S. aureus showed heterogeneity in their capacity to induce pro- and anti-inflammatory responses, suggesting that these two responses are independent of each other. Uncoupling of these responses was corroborated by selective signaling through phosphoinositol 3-kinase (PI3K)-Akt-mTOR and extracellular signal-regulated kinase (ERK) for the anti-inflammatory response and through p38 for the proinflammatory response. Uncoupling was also observed at the level of phagocytosis and phagosomal processing of S. aureus, which were required solely for the proinflammatory response. Importantly, the anti-inflammatory properties of an S. aureus isolate correlated with its ability to modulate T cell immunity. Our results suggest the presence of anti-inflammatory TLR2 ligands in the staphylococcal cell wall, whose identification may provide templates for novel immunomodulatory drugs.

Identification of key cytosolic kinases containing evolutionarily conserved kinase tyrosine-based inhibitory motifs (KTIMs).

We previously reported that SHP-1 regulates IRAK-1 activity by binding to an ITIM-like motif found within its kinase domain, which we named kinase tyrosine-based inhibitory motif (KTIM). Herein, we further investigated the presence, number, location, and evolutionary time of emergence of potential KTIMs in many cytosolic kinases, all known to play important roles in the signalling and function of immune cells. We unveil that several kinases contain potential KTIMs, mostly located within their kinase domain and appearing predominantly at the level of early vertebrates becoming highly conserved thereafter. Regarding the KTIMs that were found conserved in both vertebrates and invertebrates, we provide experimental data suggesting that such motifs may have constituted readily available sites that performed new regulatory functions as soon as their binding partners (e.g. SHP-1) appeared in vertebrates. We thus propose KTIMs as novel regulatory motifs in kinases that function through binding to SH2 domain-containing proteins such as SHP-1.

Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif.

Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKalpha/beta) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

Innate inflammatory responses to the Gram-positive bacterium Lactococcus lactis.

Lactococcus lactis is a non-pathogenic and non-colonizing Gram-positive bacterium commonly used in the dairy industry. To support the potential applications of this bacterium, such as use as an oral live vaccine, it is of interest to investigate the adjuvant properties of L. lactis. We compared the proinflammatory effects of L. lactis with two non-pathogenic Gram-negative bacteria: Escherichia coli and Salmonella typhi, a widely studied live vaccine. The gene expression profiles of chemokines induced by the three bacteria were examined in macrophages in vitro and in cells recruited into murine air-pouches in vivo. In addition, we studied the effect of co-incubating bacteria with dendritic cells (DCs) generated from mice bone marrow. We demonstrate that L. lactis exhibits proinflammatory effects, which indicates a capacity for adjuvanticity by this bacterium.