Pillon NJ, Bilan PJ, Fink LN, Klip A. Cross-talk between skeletal muscle and immune cells: muscle-derived mediators and metabolic implications.

SummaryHumans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte-derived DC in a transwell model where Gram-positive (G + ) commensals prevent Toll-like receptor-4 (TLR4)-dependent Escherichia coli-induced semimaturation in a TLR2-dependent fashion. These findings add to our understanding of the hypo-responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)-12p70, and tumour necrosis factor-a when matured with G + and Gram-negative (G -) commensals, while IL-10 production is enhanced in DC upon encountering G + commensals and reduced upon encountering G -bacteria. The Caco2 SM-induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor-b (TGF-b) and markedly reduced levels of bacteria-induced interferon-c production. Caco2 cell production of IL-8, thymic stromal lymphopoietin (TSLP) and TGF-b increases upon microbial stimulation in a strain dependent manner. TSLP and TGF-b co-operate in inducing the tolerogenic DC phenotype but other mediators might be involved.

Summary The gut microbiota is vital in the maintenance of homeostasis in the gut immune system. Its diversity and composition play major roles in relation to allergies and inflammatory bowel diseases, and administration of lactic acid bacteria (LAB), such as lactobacilli and bifidobacteria, has positive effects on these pathologies. However, the mechanisms behind the beneficial effects are largely unknown. Here we reveal divergent roles played by Toll‐like receptor‐2 (TLR2) and nucleotide‐binding oligomerization domain‐2 (NOD2) in dendritic cell (DC) recognition of LAB. Murine bone‐marrow‐derived DC lacking NOD2 produce higher levels of interleukin‐10 (IL‐10) and reduced levels of IL‐12 and tumour necrosis factor‐α (TNF‐α) in response to LAB. This indicates that peptidoglycan is partly responsible for the T helper type 1 skewing effect of certain LAB. Dendritic cells that are TLR2−/− produce less IL‐12 and TNF‐α and more IL‐10 in response to some strains of lactobacilli, while they produce more IL‐12 and less IL‐10 in response to bifidobacteria. The same tendency was found in human monocyte‐derived DC. We have previously reported that the weak IL‐12‐inducing and TNF‐α‐inducing bifidobacteria inhibit the T helper type 1 skewing effect induced by strong immunostimulatory lactobacilli. Here we show that this immunoinhibitory effect of bifidobacteria is dependent on TLR2 and independent of NOD2. Moreover, independently of the cytokine pattern induced by intact LAB, cell wall fractions of all LAB, as well as synthetic lipoproteins possess immunoinhibitory capacities in both human and murine DC. These novel findings suggest that LAB act as immunoregulators through interaction of lipoprotein with TLR2 and as immunostimulators through interaction of peptidoglycan with NOD2.

Objective: In obesity, immune cells infiltrate adipose tissue. Skeletal muscle is the major tissue of insulindependent glucose disposal, and indices of muscle inflammation arise during obesity, but whether and which immune cells increase in muscle remain unclear. Methods: Immune cell presence in quadriceps muscle of wild type mice fed high-fat diet (HFD) was studied for 3 days to 10 weeks, in CCL2-KO mice fed HFD for 1 week, and in human muscle. Leukocyte presence was assessed by gene expression of lineage markers, cyto/chemokines and receptors; immunohistochemistry; and flow cytometry. Results: After 1 week HFD, concomitantly with glucose intolerance, muscle gene expression of Ly6b, Emr1 (F4/80), Tnf, Ccl2, and Ccr2 rose, as did pro-and anti-inflammatory markers Itgax (CD11c) and Mgl2. CD11c 1 proinflammatory macrophages in muscle increased by 76%. After 10 weeks HFD, macrophages in muscle increased by 47%. Quadriceps from CCL2-KO mice on HFD did not gain macrophages and maintained insulin sensitivity. Muscle of obese, glucose-intolerant humans showed elevated CD68 (macrophage marker) and ITGAX, correlating with poor glucose disposal and adiposity. Conclusion: Mouse and human skeletal muscles gain a distinct population of inflammatory macrophages upon HFD or obesity, linked to insulin resistance in humans and CCL2 availability in mice.

Lactic acid bacteria (LAB) are abundant in the gastrointestinal tract where they continuously regulate the immune system. NK cells are potently activated by dendritic cells (DCs) matured by inflammatory stimuli, and NK cells are present in the gut epithelium and in mesenteric lymph nodes, but it is not known how NK-DC interactions are affected by the predominantly non-pathogenic LAB. We demonstrate that human DCs exposed to different strains of gut-derived LAB consistently induce proliferation, cytotoxicity and activation markers in autologous NK cells. On the contrary, strains of LAB differ greatly in their ability to induce DC-dependent IFN-gamma production by NK cells. This suggests that DCs stimulated by gut LAB may expand the pool of NK cells and increase their cytotoxic potential. Specific LAB, inducing high levels of IL-12 in DCs, may promote amplification of a type-1 response via potent stimulation of IFN-gamma production in NK cells. Combining IFN-gamma-inducing and non-inducing LAB completely abrogates DC-mediated IFN-gamma production by NK cells, and therefore LAB modulating IFN-gamma production in NK cells may be important regulators of the immune response.

A cooperative dialogue between natural killer (NK) cells and dendritic cells (DCs) has been elucidated in the last years. They help each other to acquire their complete functions, both in the periphery and in the secondary lymphoid organs. Thus, NK cells' activation by dendritic cells allows the killing of transformed or infected cells in the periphery but may also be important for the generation of adaptive immunity. Indeed, it has been shown that NK cells may play a key role in polarizing a Th1 response upon interaction with DCs exposed to microbial products. This regulatory role of DC/NK cross-talk is of particular importance at mucosal surfaces such as the intestine, where the immune system exists in intimate association with commensal bacteria such as lactic acid bacteria (LAB). We here review NK/DC interactions in the presence of gut-derived commensal bacteria and their role in bacterial strain-dependent immunomodulatory effects. We particularly aim to highlight the ability of distinct species of commensal bacterial probiotics to differently affect the outcome of DC/NK cross-talk and consequently to differently influence the polarization of the adaptive immune response.

Pseudomonas aeruginosa releases a wide array of toxins and tissue-degrading enzymes. Production of these malicious virulence factors is controlled by interbacterial communication in a process known as quorum sensing. An increasing body of evidence reveals that the bacterial signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) exhibits both quorum-sensing signalling and immune-modulating properties. Recently, yet another quorum-sensing signal molecule, the Pseudomonas quinolone signal (PQS), has been shown to affect cytokine release by mitogen-stimulated human T cells. In the present article we demonstrate that both OdDHL and PQS decrease the production of interleukin-12 (IL-12) by Escherichia coli lipopolysaccharide-stimulated bone marrow-derived dendritic cells (BM-DCs) without altering their IL-10 release. Moreover, BM-DCs exposed to PQS and OdDHL during antigen stimulation exhibit a decreased ability to induce T-cell proliferation in vitro. Collectively, this suggests that OdDHL and PQS change the maturation pattern of stimulated DCs away from a proinflammatory T-helper type I directing response, thereby decreasing the antibacterial activity of the adaptive immune defence. OdDHL and PQS thus seem to possess dual activities in the infection process: as inducers of virulence factors as well as immune-modulators facilitating the infective properties of this pathogen.

Aims/hypothesis Low-grade systemic inflammation and adipose tissue inflammatory macrophages are frequently detected in patients with obesity and type 2 diabetes. Whether inflammatory macrophages also increase in skeletal muscle of individuals with metabolic disorders remains controversial. Here, we assess whether macrophage polarisation markers in skeletal muscle of humans correlate with insulin sensitivity in obesity and type 2 diabetes. Methods Skeletal muscle biopsies were obtained from individuals of normal weight and with normal glucose tolerance (NGT), and overweight/obese individuals with or without type 2 diabetes. Insulin sensitivity was determined by euglycaemic-hyperinsulinaemic clamps. Expression of macrophage genes was analysed by quantitative RT-PCR. Results Gene expression of the inflammatory macrophage phenotype marker cluster of differentiation (CD)11c was higher in muscle of type 2 diabetes patients (p=0.0069), and correlated with HbA 1c (p=0.0139, ρ=0.48) and fasting plasma glucose (p=0.0284, ρ=0.43), but not after correction for age. Expression of TGFB1, encoding the anti-inflammatory marker TGF-β1, correlated inversely with HbA 1c (p=0.0095, ρ=−0. 50; p=0.0484, ρ=−0.50) and fasting plasma glucose (p=0. 0471, ρ=−0.39; p=0.0374, ρ=−0.52) in two cohorts, as did HbA 1c with gene expression of macrophage galactose-binding lectin (MGL) (p=0.0425, ρ=−0.51). TGFB1 expression was higher in NGT individuals than in individuals with type 2 diabetes (p=0.0303), and correlated with low fasting plasma insulin (p=0.0310, ρ=−0.42). In exercised overweight/obese individuals, expression of genes for three anti-inflammatory macrophage markers, MGL (p=0.0031, ρ=0.71), CD163 (p = 0.0268, ρ = 0.57) and mannose receptor (p = 0.0125, ρ=0.63), correlated with high glucose-disposal rate. Conclusions/interpretation Muscle expression of macrophage genes reveals a link between inflammatory macrophage markers, age and high glycaemia, whereas antiinflammatory markers correlate with low glycaemia and high glucose-disposal rate.