Downregulation of MHC Class II by Ubiquitination Is Required for the Migration of CD206+ Dendritic Cells to Skin-Draining Lymph Nodes.

Dendritic cells (DCs) are critical players in skin homeostasis. A subset of mannose receptor (CD206)-expressing monocyte-derived DCs was found in skin, and their migratory counterpart is present in skin-draining lymph nodes (sdLNs). Skin CD206+ DCs were shown to upregulate MHC class II (MHCII) progressively, raising the question of whether this feature affects their biology. In this study, we assessed the role of MHCII regulation in the development and migration of these cells in mouse models expressing differential MHCII levels. Using CD206 as a surrogate marker, we found that skin CD206+ DCs develop in an MHCII-independent manner. However, their migration to sdLNs was affected by overexpression rather than absence or lower expression of MHCII. Accordingly, B16 tumor growth was exacerbated in mice overexpressing MHCII in the absence of ubiquitination. Mechanistically, CD206+ DCs from these mice showed decreased IRF4 and CCR7 expression. LPS, which is known to promote monocyte-derived DC recruitment to sdLNs, partially improved these defects. However, GM-CSF delivery restored CD206+ DC migration by promoting IRF4 expression. Collectively, these data show that MHCII downregulation is crucial for IRF4-dependent migration of CD206+ DCs to sdLNs in health and disease.

March1 E3 Ubiquitin Ligase Modulates Features of Allergic Asthma in an Ovalbumin-Induced Mouse Model of Lung Inflammation.

Membrane-associated RING-CH-1 (March1) is a member of the March family of E3 ubiquitin ligases. March1 downregulates cell surface expression of MHC II and CD86 by targeting them to lysosomal degradation. Given the key roles of MHC class II and CD86 in T cell activation and to get further insights into the development of allergic inflammation, we asked whether March1 deficiency exacerbates or attenuates features of allergic asthma in mice. Herein, we used an acute model of allergy to compare the asthmatic phenotype of March1-deficient and -sufficient mice immunized with ovalbumin (OVA) and later challenged by intranasal instillation of OVA in the lungs. We found that eosinophilic inflammation in airways and lung tissue was similar between WT and March1-/- allergic mice, whereas neutrophilic inflammation was significant only in March1-/- mice. Airway hyperresponsiveness as well as levels of IFN-γ, IL-13, IL-6, and IL-10 was lower in the lungs of asthmatic March1-/- mice compared to WT, whereas lung levels of TNF-α, IL-4, and IL-5 were not significantly different. Interestingly, in the serum, levels of total and ova-specific IgE were reduced in March1-deficient mice as compared to WT mice. Taken together, our results demonstrate a role of March1 E3 ubiquitin ligase in modulating allergic responses.

Streptococcus suis Serotype 2 Infection Impairs Interleukin-12 Production and the MHC-II-Restricted Antigen Presentation Capacity of Dendritic Cells.

Streptococcus suis is an important swine pathogen and emerging zoonotic agent. Encapsulated strains of S. suis modulate dendritic cell (DC) functions, leading to poorly activated CD4+ T cells. However, the antigen presentation ability of S. suis-stimulated DCs has not been investigated yet. In this work, we aimed to characterize the antigen presentation profiles of S. suis-stimulated DCs, both in vitro and in vivo. Upon direct activation in vitro, S. suis-stimulated murine bone marrow-derived DCs (bmDCs) preserved their antigen capture/processing capacities. However, they showed delayed kinetics of MHC-II expression compared to lipopolysaccharide-stimulated bmDCs. Meanwhile, splenic DCs from infected mice exhibited a compromised MHC-II expression, despite an appropriate expression of maturation markers. To identify potential interfering mechanisms, Class II Major Histocompatibility Complex Transactivator (CIITA) and membrane-associated RING-CH (MARCH)1/8 transcription were studied. S. suis-stimulated DCs maintained low levels of CIITA at early time points, both in vitro and in vivo, which could limit their ability to increase MHC-II synthesis. S. suis-stimulated DCs also displayed sustained/upregulated levels of MARCH1/8, thus possibly leading to MHC-II lysosomal degradation. The bacterial capsular polysaccharide played a partial role in this modulation. Finally, interleukin (IL)-12p70 production was inhibited in splenic DCs from infected mice, a profile compatible with DC indirect activation by pro-inflammatory compounds. Consequently, these cells induced lower levels of IL-2 and TNF-α in an antigen-specific CD4+ T cell presentation assay and blunted T cell CD25 expression. It remains unclear at this stage whether these phenotypical and transcriptional modulations observed in response to S. suis in in vivo infections are part of a bacterial immune evasion strategy or rather a feature common to systemic inflammatory response-inducing agents. However, it appears that the MHC-II-restricted antigen presentation and Th1-polarizing cytokine production capacities of DCs are impaired during S. suis infection. This study highlights the potential consequences of inflammation on the type and magnitude of the immune response elicited by a pathogen.

Natural Killer Cell Functions during the Innate Immune Response to Pathogenic Streptococci.

Dendritic cells (DCs) and NK cells play a crucial role in the first phase of host defense against infections. Group B Streptococcus (GBS) and Streptococcus suis are encapsulated streptococci causing severe systemic inflammation, leading to septicemia and meningitis. Yet, the involvement of NK cells in the innate immune response to encapsulated bacterial infection is poorly characterized. Here, it was observed that these two streptococcal species rapidly induce the release of IFN-γ and that NK cells are the major cell type responsible for this production during the acute phase of the infection. Albeit S. suis capacity to activate NK cells was lower than that of GBS, these cells partially contribute to S. suis systemic infection; mainly through amplification of the inflammatory loop. In contrast, such a role was not observed during GBS systemic infection. IFN-γ release by NK cells required the presence of DCs, which in turn had a synergistic effect on DC cytokine production. These responses were mainly mediated by direct DC-NK cell contact and partially dependent on soluble factors. Though IL-12 and LFA-1 were shown to be critical in S. suis-mediated activation of the DC-NK cell crosstalk, different or redundant molecular pathways modulate DC-NK interactions during GBS infection. The bacterial capsular polysaccharides also differently modulated NK cell activation. Together, these results demonstrated a role of NK cells in the innate immune response against encapsulated streptococcal infections; yet the molecular pathways governing NK activation seem to differ upon the pathogen and should not be generalized when studying bacterial infections.

MARCH1 E3 Ubiquitin Ligase Dampens the Innate Inflammatory Response by Modulating Monocyte Functions in Mice.

Ubiquitination was recently identified as a central process in the pathogenesis and development of numerous inflammatory diseases, such as obesity, atherosclerosis, and asthma. Treatment with proteasomal inhibitors led to severe side effects because ubiquitination is heavily involved in a plethora of cellular functions. Thus, new players regulating ubiquitination processes must be identified to improve therapies for inflammatory diseases. In addition to their role in adaptive immunity, endosomal MHC class II (MHCII) molecules were shown to modulate innate immune responses by fine tuning the TLR4 signaling pathway. However, the role of MHCII ubiquitination by membrane associated ring-CH-type finger 1 (MARCH1) E3 ubiquitin ligase in this process remains to be assessed. In this article, we demonstrate that MARCH1 is a key inhibitor of innate inflammation in response to bacterial endotoxins. The higher mortality of March1-/- mice challenged with a lethal dose of LPS was associated with significantly stronger systemic production of proinflammatory cytokines and splenic NK cell activation; however, we did not find evidence that MARCH1 modulates LPS or IL-10 signaling pathways. Instead, the mechanism by which MARCH1 protects against endotoxic shock rests on its capacity to promote the transition of monocytes from Ly6CHi to Ly6C+/- Moreover, in competitive bone marrow chimeras, March1-/- monocytes and polymorphonuclear neutrophils outcompeted wild-type cells with regard to bone marrow egress and homing to peripheral organs. We conclude that MARCH1 exerts MHCII-independent effects that regulate the innate arm of immunity. Thus, MARCH1 might represent a potential new target for emerging therapies based on ubiquitination reactions in inflammatory diseases.

Immune-responsiveness of CD4+ T cells during Streptococcus suis serotype 2 infection.

The pathogenesis of Streptococcus suis infection, a major swine and human pathogen, is only partially understood and knowledge on the host adaptive immune response is critically scarce. Yet, S. suis virulence factors, particularly its capsular polysaccharide (CPS), enable this bacterium to modulate dendritic cell (DC) functions and potentially impair the immune response. This study aimed to evaluate modulation of T cell activation during S. suis infection and the role of DCs in this response. S. suis-stimulated total mouse splenocytes readily produced TNF-α, IL-6, IFN-γ, CCL3, CXCL9, and IL-10. Ex vivo and in vivo analyses revealed the involvement of CD4+ T cells and a Th1 response. Nevertheless, during S. suis infection, levels of the Th1-derived cytokines TNF-α and IFN-γ were very low. A transient splenic depletion of CD4+ T cells and a poor memory response were also observed. Moreover, CD4+ T cells secreted IL-10 and failed to up-regulate optimal levels of CD40L and CD69 in coculture with DCs. The CPS hampered release of several T cell-derived cytokines in vitro. Finally, a correlation was established between severe clinical signs of S. suis disease and impaired antibody responses. Altogether, these results suggest S. suis interferes with the adaptive immune response.

Group B Streptococcus Induces a Robust IFN-γ Response by CD4(+) T Cells in an In Vitro and In Vivo Model.

Group B Streptococcus (GBS) serotype III causes life-threatening infections. Cytokines have emerged as important players for the control of disease, particularly IFN-γ. Although potential sources of this cytokine have been proposed, no specific cell line has ever been described as a leading contributor. In this study, CD4(+) T cell activation profiles in response to GBS were evaluated through in vivo, ex vivo, and in vitro approaches. Total splenocytes readily produce a type 1 proinflammatory response by releasing IFN-γ, TNF-α, and IL-6 and actively recruit T cells via chemokines like CXCL9, CXCL10, and CCL3. Responding CD4(+) T cells differentiate into Th1 cells producing large amounts of IFN-γ, TNF-α, and IL-2. In vitro studies using dendritic cell and CD4(+) T cell cocultures infected with wild-type GBS or a nonencapsulated mutant suggested that GBS capsular polysaccharide, one of the major bacterial virulence factors, differentially modulates surface expression of CD69 and IFN-γ production. Overall, CD4(+) T cells are important producers of IFN-γ and might thus influence the course of GBS infection through the expression balance of this cytokine.

Tollip-induced down-regulation of MARCH1.

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA(+) HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.

MARCH1 down-regulation in IL-10-activated B cells increases MHC class II expression.

IL-10 is vastly studied for its anti-inflammatory properties on most immune cells. However, it has been reported that IL-10 activates B cells, up-regulates their MHC class II molecules and prevents apoptosis. As MARCH1 was shown to be responsible for the intracellular sequestration of MHC class II molecules in dendritic cells and monocytes in response to IL-10, we set out to clarify the role of this ubiquitin ligase in B cells. Here, we demonstrate in mice that splenic follicular B cells represent the major cell population that up-regulate MHC II molecules in the presence of IL-10. Activation of these cells through TLR4, CD40 or the IL-10 receptor caused the down-regulation of MARCH1 mRNA. Accordingly, B cells from MARCH1-deficient mice do not up-regulate I-A(b) in response to IL-10. In all, our results demonstrate that IL-10 can have opposite effects on MARCH1 regulation in different cell types.