Several research works have demonstrated that beneficial microbes with the capacity to modulate the mucosal immune system (immunobiotics) are an interesting alternative to improve the outcome of bacterial and viral respiratory infections. Among the immunobiotic strains with the capacity to beneficially modulate respiratory immunity, Lactobacillus rhamnosus CRL1505 has outstanding properties. Although we have significantly advanced in demonstrating the capacity of L. rhamnosus CRL1505 to improve resistance against respiratory infections as well as in the cellular and molecular mechanisms involved in its beneficial activities, the potential protective ability of this strain or its immunomodulatory cellular fractions in the context of a secondary bacterial pneumonia has not been addressed before. In this work, we demonstrated that the nasal priming with non-viable L. rhamnosus CRL1505 or its purified peptidoglycan differentially modulated the respiratory innate antiviral immune response triggered by toll-like receptor 3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that peptidoglycan from L. rhamnosus CRL1505 significantly improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-β+ alveolar macrophages with the consequent increases of IFN-γ, IL-10, and IFN-β in the respiratory tract. Our results also showed that the increase of these three cytokines is necessary to achieve protection against respiratory superinfection since each of them are involved in different aspect of the secondary pneumococcal pneumonia that have to be controlled in order to reduce the severity of the infectious disease: lung pneumococcal colonization, bacteremia, and inflammatory-mediated lung tissue injury.

This work demonstrates the reduction of TLR3-mediated intestinal tissue injury by immunobiotic lactobacilli through the modulation of intraepithelial lymphocytes response. It is a step forward in the understanding of the cellular mechanisms involved in the antiviral capabilities of immunobiotic strains.

Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

Intravenous chemotherapy has poor access to metastatic lymph nodes (LNs) and is limited by short-lived drug concentrations. Here, we describe the administration of chemotherapy via the lymphatic network as a new concept for the prevention and treatment of metastatic LNs. A metastatic LN can be treated by the injection of drugs into an upstream LN, either the sentinel LN (SLN) or another upstream LN. In a mouse model, tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis to the proper axillary LN (PALN). Two routes were used for drug delivery to the PALN, namely from the SiLN and from the accessory axillary LN (AALN). We found that tumor masses were formed in lymphatic vessels between the SiLN and PALN. The flow of fluorescent solution injected into the SiLN towards the PALN decreased with tumor mass formation. Delivery from the AALN (free of metastatic tumor cells) to the PALN was identified as an alternative route. Intranodal injection can deliver high concentrations of drugs to secondary metastatic LNs. The study advocates a new concept for the prevention and treatment of metastatic lymph nodes whereby drugs injected into upstream lymph nodes can reach metastatic lymph nodes via the lymphatic network.

Systemic administration of drugs into the blood circulation is standard treatment for prevention of metastasis. However, systemic delivery cannot maintain sufficiently high concentrations of anticancer drugs in lymph nodes (LN). Here, we show that giving cisplatin (CDDP) using a lymphatic drug delivery system (LDDS) has the potential to treat false‐negative metastatic LN. We found that in MXH10/Mo‐lpr/lpr mice, which develop systemic swelling of LN up to 10 mm in diameter, accumulation of indocyanine green (ICG), which has a similar molecular weight to CDDP, in a target LN was greater for lymphatic delivery of ICG than for systemic (i.v.) administration. Furthermore, CDDP administration with a LDDS inhibited tumor growth in false‐negative metastatic LN and produced fewer adverse effects than systemically given CDDP. We anticipate that drug delivery using a LDDS will, in time, replace systemic chemotherapy for the treatment of false‐negative metastatic LN.

We previously established a clonal porcine intramuscular preadipocyte (PIP) line and we were able to establish a protocol to obtain functional mature adipocytes from PIP cells. We hypothesized that both PIP cells and mature adipocytes are likely to be useful in vitro tools for increasing our understanding of immunobiology of adipose tissue, and for the selection and study of immunoregulatory probiotics (immunobiotics) able to modulate adipocytes immune responses. In this study, we investigated the immunobiology of PIP cells and mature adipocytes in relation to their response to TNF-α stimulation. In addition, we evaluated the possibility that immunobiotic microorganisms modify adipogenesis and immune functions of porcine adipose tissue through Peyer’s patches (PPs) immune-competent cells. We treated the porcine PPs immune cells with different probiotic strains; and we evaluated the effect of conditioned media from probiotic-stimulated immune cells in PIP cells and mature adipocytes. The Lactobacillus GG and L. gasseri TMC0356 showed remarkable effects, and were able to significantly reduce the expression of pro-inflammatory factors and negative regulators (A20, Bcl-3, and MKP-1) in adipocytes challenged with TNF-α. The results of this study demonstrated that the evaluation of IL-6, and MCP-1 production, and A20 and Bcl-3 down-regulation in TNF-α-challenged adipocytes could function as biomarkers to screen and select potential immunobiotic strains. Taking into consideration that several in vivo and in vitro studies clearly demonstrated the beneficial effects of Lactobacillus GG and L. gasseri TMC0356 in adipose inflammation, the results presented in this work indicate that the PIP cells and porcine adipocytes could be used for the screening and the selection of new immunobiotic strains with the potential to functionally modulate adipose inflammation when orally administered.

Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the ‘PPAR signaling’ and ‘insulin resistance’ pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of ′GPCR ligand-binding′, ‘cell chemotaxis’, ‘blood coagulation and complement’, ‘metabolism of lipid and lipoproteins’, ‘regulation of lipid metabolism by PPARA’, and ‘lipid digestion, mobilization and transport’ pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10 , and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2 , and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.