Philippe Pouliot

Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen

It is unclear how Th2 immunity is induced in response to allergens like house dust mite (HDM). Here, we show that HDM inhalation leads to the TLR4/MyD88-dependent recruitment of IL-4 competent basophils and eosinophils, and of inflammatory DCs to the draining mediastinal nodes. Depletion of basophils only partially reduced Th2 immunity, and depletion of eosinophils had no effect on the Th2 response. Basophils did not take up inhaled antigen, present it to T cells, or express antigen presentation machinery, whereas a population of FceRI+ DCs readily did. Inflammatory DCs were necessary and sufficient for induction of Th2 immunity and features of asthma, whereas basophils were not required. We favor a model whereby DCs initiate and basophils amplify Th2 immunity to HDM allergen.

Interleukin-1α controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33

IL-1α promotes a cascade of cytokine production from epithelial cells culminating in Th2 immunity to house dust mite allergens.

An Unexpected Role for Uric Acid as an Inducer of T Helper 2 Cell Immunity to Inhaled Antigens and Inflammatory Mediator of Allergic Asthma

Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation.

The unfolded-protein-response sensor IRE-1α regulates the function of CD8α + dendritic cells

The role of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in homeostasis of the immune system is incompletely understood. Here we found that dendritic cells (DCs) constitutively activated the UPR sensor IRE-1α and its target, the transcription factor XBP-1, in the absence of ER stress. Loss of XBP-1 in CD11c+ cells led to defects in phenotype, ER homeostasis and antigen presentation by CD8α+ conventional DCs, yet the closely related CD11b+ DCs were unaffected. Whereas the dysregulated ER in XBP-1-deficient DCs resulted from loss of XBP-1 transcriptional activity, the phenotypic and functional defects resulted from regulated IRE-1α-dependent degradation (RIDD) of mRNAs, including those encoding CD18 integrins and components of the major histocompatibility complex (MHC) class I machinery. Thus, a precisely regulated feedback circuit involving IRE-1α and XBP-1 controls the homeostasis of CD8α+ conventional DCs.

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

Background Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously. Methodology/Principal Findings Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohns disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa. Conclusions/Significance Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.

Aspergillus fumigatus Induces Immunoglobulin E-Independent Mast Cell Degranulation

BACKGROUND Pulmonary colonization by Aspergillus fumigatus in chronic lung disease is associated with progressive decline in lung function even in the absence of specific allergic response. We hypothesized that A. fumigatus contributes to this decline by inducing pulmonary mast cell degranulation even in the absence of antigen-specific immunoglobulin E (IgE). Therefore, we investigated whether A. fumigatus can induce mast cell degranulation independently of IgE. METHODS We studied the interactions of Aspergillus species with mast cells in the absence of IgE in vitro with use of scanning electron microscopy. The extent of mast cell degranulation was quantified by measuring the release of beta-hexosaminidase. RESULTS Mature A. fumigatus hyphae induced mast cell degranulation in the absence of IgE. Hyphae of Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans induced much less mast cell degranulation. Mast cell degranulation required direct contact with mature A. fumigatus hyphae, and was not induced by conidia or immature hyphae. Killed hyphae induced significant degranulation, whereas live hyphae from mutants deficient in the fungal development regulators StuA and MedA induced very little degranulation. CONCLUSIONS Factors expressed on the surface of mature A. fumigatus hyphae that are controlled by StuA and MedA induce mast cell degranulation in the absence of IgE.

Myeloid-Related Proteins Rapidly Modulate Macrophage Nitric Oxide Production during Innate Immune Response

S100A8 and S100A9 are intracellular calcium-binding proteins produced by myeloid cells that promote neutrophil/monocyte recruitment at inflamed tissues by enhancing attachment to endothelial cells. Although the intracellular functions of these proteins, i.e., myeloid-related proteins (MRP)-8 and MRP-14, are not completely understood, these proteins exhibit prominent extracellular cytokine-like functions and are considered reliable markers of inflammation in diverse diseases. As S100A8 and S100A9 have been reported to be rapidly released in response to components derived from infectious agents, we hypothesized that they play an important role in the modulation of key microbicidal phagocyte functions. In this study, we report for the first time that MRPs are powerful inducers of NO production by murine macrophages (Mφ). This increase in NO production was linked to an increased inducible NO synthase expression both at gene and protein level. This induction was concomitant with an important phosphorylation of SAPK/JNK, but also of MEK and ERK kinases. Upon stimulation with MRPs, NF-κB was rapidly translocated to the nucleus (30 min). When Mφ were treated concomitantly with IFN-γ, another activator of Mφ functions, we observed a strong synergy in NO production, synergy that resulted from the engagement of exclusive signaling pathways: SAPK/JNK, ERK and NF-κB were involved in signaling of MRPs, whereas IFN-γ uses the JAK/STAT pathway. This suggests that the synergy results from interactions of transcription factors in the promoter region. Finally, we observed this effect to be dependent on TLR4. Collectively, our study unravels the importance of MRPs as potent new inducers of Mφ NO production.

Allosteric competitive inactivation of hematopoietic CSF-1 signaling by the viral decoy receptor BARF1

Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor–binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid. BARF1 locks dimeric hCSF-1 into an inactive conformation, rendering it unable to signal via its cognate receptor on human monocytes. This reveals a new functional role for hCSF-1 cooperativity in signaling. We propose a new viral strategy paradigm featuring an allosteric decoy receptor of the competitive type, which couples efficient sequestration and inactivation of the host growth factor to abrogate cooperative assembly of the cognate signaling complex.

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.

Generation and evaluation of A2-expressing Lactococcus lactis live vaccines against Leishmania donovani in BALB/c mice

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. As current treatments are insufficient, the development of an effective vaccine is a priority. This study generated and assessed the efficacy of Leishmania vaccines engineered from the non-colonizing, non-pathogenic Gram-positive bacterium Lactococcus lactis. A truncated, codon-optimized version of the A2 antigen from Leishmania donovani was engineered for expression in Lactococcus lactis in three different subcellular compartments: in the cytoplasm, secreted outside the cell or anchored to the cell wall. These three A2-expressing Lactococcus lactis strains were tested for their ability to generate A2-specific immune responses and as live vaccines against visceral Leishmania donovani infection in BALB/c mice. Subcutaneous immunization with live Lactococcus lactis expressing A2 anchored to the cell wall effectively induced high levels of antigen-specific serum antibodies. It was demonstrated that Lactococcus lactis-based vaccines are a feasible approach in the generation of live vaccines against leishmaniasis. The Lactococcus lactis strains generated in this study provide an excellent foundation for further studies on live bacterial vaccines against leishmaniasis and other pathogens.