Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice.

The intestinal microbiota influences immune maturation during childhood, and is implicated in early-life allergy development. However, to directly study intestinal microbes and gut immune responses in infants is difficult. To investigate how different types of early-life gut microbiota affect immune development, we collected fecal samples from children with different allergic heredity (AH) and inoculated germ-free mice. Immune responses and microbiota composition were evaluated in the offspring of these mice. Microbial composition in the small intestine, the cecum and the colon were determined by 16S rRNA sequencing. The intestinal microbiota differed markedly between the groups of mice, but only exposure to microbiota associated with AH and known future allergy in children resulted in a T helper 17 (Th17)-signature, both systemically and in the gut mucosa in the mouse offspring. These Th17 responses could be signs of a particular microbiota and a shift in immune development, ultimately resulting in an increased risk of allergy.

Macrophage control of phagocytosed mycobacteria is increased by factors secreted by alveolar epithelial cells through nitric oxide independent mechanisms.

Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM). We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms.

Lactobacilli regulate Staphylococcus aureus 161:2-induced pro-inflammatory T-cell responses in vitro.

There seems to be a correlation between early gut microbiota composition and postnatal immune development. Alteration in the microbial composition early in life has been associated with immune mediated diseases, such as autoimmunity and allergy. We have previously observed associations between the presence of lactobacilli and Staphylococcus (S.) aureus in the early-life gut microbiota, cytokine responses and allergy development in children. Consistent with the objective to understand how bacteria modulate the cytokine response of intestinal epithelial cell (IEC) lines and immune cells, we exposed IEC lines (HT29, SW480) to UV-killed bacteria and/or culture supernatants (-sn) from seven Lactobacillus strains and three S. aureus strains, while peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated by bacteria-sn or with bacteria conditioned IEC-sn. Although the overall IEC response to bacterial exposure was characterized by limited sets of cytokine and chemokine production, S. aureus 161:2-sn induced an inflammatory response in the IEC, characterized by CXCL1/GROα and CXCL8/IL-8 production, partly in a MyD88-dependent manner. UV-killed bacteria did not induce a response in the IEC line, and a combination of both UV-killed bacteria and the bacteria-sn had no additive effect to that of the supernatant alone. In PBMC, most of the Lactobacillus-sn and S. aureus-sn strains were able to induce a wide array of cytokines, but only S. aureus-sn induced the T-cell associated cytokines IL-2, IL-17 and IFN-γ, independently of IEC-produced factors, and induced up regulation of CTLA-4 expression and IL-10 production by T-regulatory cells. Notably, S. aureus-sn-induced T-cell production of IFN- γ and IL-17 was down regulated by the simultaneous presence of any of the different Lactobacillus strains, while the IEC CXCL8/IL-8 response was unaltered. Thus these studies present a possible role for lactobacilli in induction of immune cell regulation, although the mechanisms need to be further elucidated.

Alveolar epithelial cells are critical in protection of the respiratory tract by secretion of factors able to modulate the activity of pulmonary macrophages and directly control bacterial growth.

The respiratory epithelium is a physical and functional barrier actively involved in the clearance of environmental agents. The alveolar compartment is lined with membranous pneumocytes, known as type I alveolar epithelial cells (AEC I), and granular pneumocytes, type II alveolar epithelial cells (AEC II). AEC II are responsible for epithelial reparation upon injury and ion transport and are very active immunologically, contributing to lung defense by secreting antimicrobial factors. AEC II also secrete a broad variety of factors, such as cytokines and chemokines, involved in activation and differentiation of immune cells and are able to present antigen to specific T cells. Another cell type important in lung defense is the pulmonary macrophage (PuM). Considering the architecture of the alveoli, a good communication between the external and the internal compartments is crucial to mount effective responses. Our hypothesis is that being in the interface, AEC may play an important role in transmitting signals from the external to the internal compartment and in modulating the activity of PuM. For this, we collected supernatants from AEC unstimulated or stimulated in vitro with lipopolysaccharide (LPS). These AEC-conditioned media were used in various setups to test for the effects on a number of macrophage functions: (i) migration, (ii) phagocytosis and intracellular control of bacterial growth, and (iii) phenotypic changes and morphology. Finally, we tested the direct effect of AEC-conditioned media on bacterial growth. We found that AEC-secreted factors had a dual effect, on one hand controlling bacterial growth and on the other hand increasing macrophage activity.

The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems.

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.

Impact of toll-like receptor 2 deficiency on immune responses to mycobacterial antigens.

In the present study, we addressed the question of whether Toll-like receptor 2 (TLR2)-mediated innate immunity can contribute to the development of acquired immune responses. We immunized TLR2(-/-) and wild-type (WT) mice three times subcutaneously with the mycobacterial antigen (Ag19kDa) (a TLR2 ligand) or Ag85A (not a TLR2 ligand). One week after the last immunization, sera and spleens were collected. To evaluate cellular responses, we measured gamma interferon (IFN-γ) after in vitro restimulation of spleen cells with antigen alone or antigen-pulsed bone marrow-derived macrophages (BMM(Ag)) or pulmonary macrophages (PuM(Ag)). Antibody responses were comparable in the two mouse strains, but we observed differences in the cellular responses. Recall responses to Ag85A were similar in the two strains, but responses to Ag19kDa given alone or presented by BMM or PuM were lower in TLR2(-/-) than in WT mice. The largest differences in cellular responses were observed when Ag19kDa was presented by PuM. To understand this, we analyzed phenotypic and functional differences between BMM and PuM upon stimulation with various ligands. Generally, PuM had a lower response to the TLR2 ligand Pam(3)Cys-Ser-(Lys)(4) trihydrochloride and to anti-CD40 than BMM, as measured by cytokine secretion and upregulation of costimulatory molecules. This might provide a partial explanation for the lower capacity of PuM when pulsed with Ag19kDa, also a TLR2 ligand. Altogether, our results revealed weaknesses in the T cell and antigen-presenting cell (APC) compartments of the Ag19kDa-immunized TLR2(-/-) mice but indicated that specific immune responses could be generated in the absence of TLR2 regardless of the characteristics of the antigen used.

Dietary fish oil decreases secretion of T helper (Th) 1-type cytokines by a direct effect on murine splenic T cells but enhances secretion of a Th2-type cytokine by an effect on accessory cells.

Dietary fish oil is considered to have anti-inflammatory effects based primarily on its effects on T-cell proliferation and IL-2 secretion. Its effects on the secretion of T helper (Th) 1-type cytokines vary and few studies have examined its effects on the secretion of Th2-type cytokines. In the present study, we examined the effects of dietary fish oil on the secretion of Th1 and Th2-type cytokines by splenocytes and the mechanism by which dietary fish oil affects Th2-type cytokine secretion. Mice were fed diets supplemented with 18 % fish oil (w/w) +2 % maize oil or 20 % maize oil for 6 weeks. Spleen cells, isolated splenic T cells and accessory cells (splenocytes depleted of T cells) were stimulated with anti-CD3/anti-CD28. The secretion of interferon (IFN)-gamma, TNF-alpha, IL-4 and IL-10 was measured by ELISA. Dietary fish oil decreased the secretion of IFN-gamma and TNF-alpha by total splenocytes and isolated T cells. In contrast, dietary fish oil increased the secretion of IL-4 by total splenocytes but had no effect on IL-4 secretion by isolated T cells. When isolated T cells were cultured with CD11b+ cells (mainly macrophages), cells from mice fed the fish oil diet secreted more IL-4 than cells from mice fed the maize oil diet. These results demonstrate that dietary fish oil directs cytokine secretion by splenocytes towards a Th2 phenotype and that the effects of dietary fish oil on the secretion of a Th2-type cytokine are mediated by its effect on CD11b+ accessory cells.

The effects of omega-3 polyunsaturated fatty acids on TNF-alpha and IL-10 secretion by murine peritoneal cells in vitro.

Omega-3 polyunsaturated fatty acids (PUFA) affect immune response, partly by affecting cytokine secretion. Omega-3 PUFA decrease tumor necrosis factor (TNF)-alpha secretion by RAW 264.7 macrophages but increase TNF-alpha secretion by primary elicited peritoneal macrophages in vitro. In this study, the effects of omega-3 and omega-6 PUFA on lipopolysaccharide induced TNF-alpha and interleukin (IL)-10 secretion by murine primary resident and elicited peritoneal macrophages and by RAW 264.7 macrophages, were examined in vitro using an enzyme-linked immunosorbent assay. In addition, the effects of dietary omega-3 PUFA on the number of cells secreting these cytokines were examined with enzyme-linked immunospot assay. All cell types secreted more TNF-alpha but similar amounts of IL-10 when incubated with the omega-3 PUFA, eicosapentaenoic acid or docosahexaenoic acid, compared with that when incubated with the omega-6 PUFA, linoleic acid or arachidonic acid. Dietary fish oil did not affect the number of TNF-alpha secreting resident peritoneal macrophages but decreased the number of macrophages secreting IL-10 ex vivo. These results show that dietary omega-3 PUFA and omega-3 PUFA added to cells in vitro increase TNF-alpha secretion by resident peritoneal macrophages, probably by a direct effect on the cells. In contrast, omega-3 PUFA did not affect IL-10 secretion by the cells but decreased the number of cells secreting IL-10 ex vivo, possibly by affecting cell recruitment, maturation or proliferation.

Dietary fish oil increases the number of splenic macrophages secreting TNF-alpha and IL-10 but decreases the secretion of these cytokines by splenic T cells from mice.

Dietary fish oil has immunomodulatory effects that are partly mediated by its effects on cytokine secretion. In this paper, we examine whether dietary fish oil has different effects on cytokine secretion by T cells and macrophages. Female BalbC mice were fed diets supplemented with 18% fish oil + 2% corn oil or 20% corn oil. Concanavalin A (ConA)- and LPS-induced TNF-alpha and IL-10 secretion by splenocytes was examined using ELISA. Dietary fish oil decreased ConA induced-, but increased LPS-induced, TNF-alpha and IL-10 secretion by total murine splenocytes. Dietary fish oil increased the number of splenocytes secreting TNF-alpha and IL-10, following stimulation with LPS, by 123 and 38%, respectively, but did not affect cytokine secretion by each cell, as determined using enzyme-linked immunospot. Spleens from mice fed the fish oil diet had over 2-fold higher proportion of macrophages with high expression of CD11b than spleens from mice fed the corn oil diet. In addition, fish oil increased the proportion of total and CD11b(+) splenocytes that expressed the LPS receptor complex molecules, CD14 and toll-like receptor (TLR)4/myeloid differentiation factor-2 (MD-2), by 85 and 28%, respectively. The increased proportion of macrophages expressing the LPS receptor complex molecules, CD14 and TLR4/MD-2, in spleens from mice fed the fish oil diet may explain the increased number of cells that secreted the cytokines after LPS stimulation.

Dietary fish oil increases tumor necrosis factor secretion but decreases interleukin-10 secretion by murine peritoneal macrophages.

Dietary fish oil has immunomodulatory effects that are mediated in part by its effects on cytokines. Secretion of the inflammatory and the anti-inflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-10 by murine resident peritoneal macrophages was monitored after ex vivo stimulation with lipopolysaccharide. Macrophages were obtained from mice fed a corn oil diet containing 200 g/kg corn oil or a fish oil diet containing 180 g/kg fish oil and 20 g/kg corn oil. Dietary fish oil increased secretion of the proinflammatory cytokine, TNF, but decreased secretion of the anti-inflammatory cytokine, IL-10. The cytokines appeared in the medium after 1.5 h and peaked at 6-12 h. Neutralizing antibodies against TNF and IL-10 had little effect on secretion of the other cytokine, indicating that the effects of fish oil on TNF and IL-10 secretion by these cells are independent of one another. Furthermore, although inhibiting prostaglandin production enhanced TNF secretion by macrophages from mice fed the corn oil diet, it did not affect IL-10 secretion by macrophages in this group. Blocking leukotriene B(4) production also did not affect IL-10 secretion in macrophages from mice fed a nonpurified diet. These results demonstrate that fish oil has an overall pro-inflammatory effect given its effects on secretion of both inflammatory and anti-inflammatory cytokines by resident peritoneal macrophages.