Specific preadaptations of Rhodococcus equi cooperate with its Virulence-associated protein A during macrophage infection.

Gram-positive Rhodococcus equi (Prescotella equi) is a lung pathogen of foals and immunocompromised humans. Intra-macrophage multiplication requires production of the bacterial Virulence-associated protein A (VapA) which is released into the phagosome lumen. VapA pH-neutralizes intracellular compartments allowing R. equi to multiply in an atypical macrophage phagolysosome. Here, we show that VapA does not support intra-macrophage growth of several other bacterial species demonstrating that only few bacteria have the specific preadaptations needed to profit from VapA. We show that the closest relative of R. equi, environmental Rhodococcus defluvii (Prescotella defluvii), does not multiply in macrophages at 37°C even when VapA is present because of its thermosensitivity but it does so once the infection temperature is lowered providing rare experimental evidence for 'thermal restriction'. Using growth experiments with isolated macrophage lysosomes and modified infection schemes we provide evidence that R. equi resists the attack by phagolysosome contents at low pH for several hours. During this time, R. equi produces and secretes VapA which enables it to grow at the expense of lysosome constituents. We present arguments that, under natural infection conditions, R. equi is VapA-less during the initial encounter with the host. This has important implications for vaccine development.

Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease.

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling.

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model.

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

NOX2 Deficiency Permits Sustained Survival of S. aureus in Macrophages and Contributes to Severity of Infection.

Although the crucial role of professional phagocytes for the clearance of S. aureus infections is well-established, several studies indicate an adverse role of leukocytes in the dissemination of S. aureus during infection. Since only little is known about macrophages in this context, we analyzed the role of macrophages, and in particular reactive oxygen species deficiency, for the seeding of S. aureus metastases. Infection of bone marrow-derived macrophages (BMDM) with S. aureus revealed that NADPH oxidase 2 (NOX2-) deficient, but not NOX1- or NOX4-deficient, BMDM failed to clear intracellular S. aureus. Despite of larger intracellular bacterial burden, NOX2-deficient BMDM showed significantly improved survival. Intravenous injection of mice with in vitro-infected BMDMs carrying intracellular viable S. aureus led to higher bacterial loads in kidney and liver of mice compared to injection with plain S. aureus. An even higher frequency of liver abscesses was observed in mice infected with S. aureus-loaded nox2-/- BMDM. Thus, the improved intracellular survival of S. aureus and improved viability of NOX2-deficient BMDM is associated with an aggravated metastatic dissemination of S. aureus infection. A combination of vancomycin and the intracellularly active antibiotic rifampicin led to complete elimination of S. aureus from liver within 48 h, which was not achieved with vancomycin treatment alone, underscoring the impact of intracellular S. aureus on the course of disease. The results of our study indicate that intracellular S. aureus carried by macrophages are sufficient to establish a systemic infection. This suggests the inclusion of intracellularly active antibiotics in the therapeutic regimen of invasive S. aureus infections, especially in patients with NADPH oxidase deficiencies such as chronic granulomatous disease.

Ceramide Synthase 5 Deficiency Aggravates Dextran Sodium Sulfate-Induced Colitis and Colon Carcinogenesis and Impairs T-Cell Activation.

Ceramide synthase 5 is one of six enzymes that catalyze the production of ceramides from sphingosine or sphinganine. Ceramides are important components of cell membranes and act as signaling molecules. Previously it has been shown that ceramide synthase 6 and 2 influence colitis in several animal models with sometimes opposite effects. Here, we investigated the disease course of dextran sodium sulfate-induced acute colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer in mice with global ceramide synthase 5 knockout (CerS5-ko) or with ceramide synthase 5 knockout restricted to the colon epithelium (CerS5fl/fl VilCre). We monitored disease development and analyzed colon barrier function as well as the immune cell status in these mice. CerS5-ko mice but not CerS5fl/fl-VilCre mice were more susceptible to acute and chronic inflammation. However, the cell barrier function of colon epithelial cells was not disturbed by downregulation of ceramide synthase 5. Instead, untreated CerS5-ko mice displayed reduced numbers of CD3+ immune cells in the spleen, colon, and blood, especially of intraepithelial CD8+ T-cells, which was not obvious in CerS5fl/fl Vil Cre mice. Reduced T-cell number in colon tissue of CerS5-ko mice was accompanied by a reduced expression of IL-1ß, IFNγ, and IL-4. In vitro investigations revealed that knockdown of ceramide synthase 5 in T-cells impaired T-cell activation. In summary, we show that CerS5-ko mice were more susceptible to dextran sodium sulfate-induced colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer. A reduced number of T-cells in the colon epithelium that was already the case in untreated CerS5-ko mice might have contributed to this effect.

ATM activity in T cells is critical for immune surveillance of lymphoma in vivo.

The proximal DNA damage response kinase ATM is frequently inactivated in human malignancies. Germline mutations in the ATM gene cause Ataxia-telangiectasia (A-T), characterized by cerebellar ataxia and cancer predisposition. Whether ATM deficiency impacts on tumor initiation or also on the maintenance of the malignant state is unclear. Here, we show that Atm reactivation in initially Atm-deficient B- and T cell lymphomas induces tumor regression. We further find a reduced T cell abundance in B cell lymphomas from Atm-defective mice and A-T patients. Using T cell-specific Atm-knockout models, as well as allogeneic transplantation experiments, we pinpoint impaired immune surveillance as a contributor to cancer predisposition and development. Moreover, we demonstrate that Atm-deficient T cells display impaired proliferation capacity upon stimulation, due to replication stress. Altogether, our data indicate that T cell-specific restoration of ATM activity or allogeneic hematopoietic stem cell transplantation may prevent lymphomagenesis in A-T patients.

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.

A major function of macrophages during infection is initiation of the proinflammatory response, leading to the secretion of cytokines that help to orchestrate the immune response. Here, we identify reactive oxygen species (ROS) as crucial mediators of proinflammatory signaling leading to cytokine secretion in Listeria monocytogenes-infected macrophages. ROS produced by NADPH oxidases (Noxes), such as Nox2, are key components of the macrophage response to invading pathogens; however, our data show that the ROS that mediated proinflammatory signaling were produced by mitochondria (mtROS). We identified the inhibitor of κB (IκB) kinase (IKK) complex regulatory subunit NEMO [nuclear factor κB (NF-κB) essential modulator] as a target for mtROS. Specifically, mtROS induced intermolecular covalent linkage of NEMO through disulfide bonds formed by Cys54 and Cys347, which was essential for activation of the IKK complex and subsequent signaling through the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and NF-κB pathways that eventually led to the secretion of proinflammatory cytokines. We thus identify mtROS-dependent disulfide linkage of NEMO as an essential regulatory step of the proinflammatory response of macrophages to bacterial infection.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

The ß2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes.

The intracellular pathogen Listeria monocytogenes (L.m.) is targeted by the autophagic machinery, but the molecular mechanisms involved and consequences for anti-listerial immunity remain enigmatic. Here, we demonstrate that L.m. infection of macrophages in vivo exclusively evokes LC3-associated phagocytosis (LAP), but not canonical autophagy, and that targeting of L.m. by LAP is required for anti-listerial immunity. The pathway leading to LAP induction in response to L.m. infection emanates from the ß2 integrin Mac-1 (CR3, integrin αMß2), a receptor recognizing diverse microbial ligands. Interaction of L.m. with Mac-1 induces acid sphingomyelinase-mediated changes in membrane lipid composition that facilitate assembly and activation of the phagocyte NAPDH oxidase Nox2. Nox2-derived reactive oxygen species then trigger LC3 recruitment to L.m.-containing phagosomes by LAP. By promoting fusion of L.m.-containing phagosomes with lysosomes, LAP increases exposure of L.m. to bactericidal acid hydrolases, thereby enhancing anti-listerial activity of macrophages and immunity of mice.

Using Antigen-Specific B Cells to Combine Antibody and T Cell-Based Cancer Immunotherapy.

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1-specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730-43. ©2017 AACR.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates. CONCLUSION: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver-directed gene therapy using AAV vectors and showed in a proof-of-principle study how this virus-host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252-265).

Toll-Like Receptor 2, Toll-Like Receptor 4, Myeloid Differentiation Response Gene 88, and Toll-IL-1 Receptor Domain-Containing Adaptor-Inducing Interferon-γ (TRIF) Selectively Regulate Susceptibility of P0106-125-Induced Murine Experimental Autoimmune Neuritis.

The functional relevance of the innate immune system has not yet been dissected in P0106-125-induced murine experimental autoimmune neuritis. Therefore, the role of Toll-like receptor (TLR) 2, TLR4, myeloid differentiation response gene 88, and Toll-IL-1 receptor domain-containing adaptor-inducing interferon-γ (TRIF), factors critically involved in the TLR signaling pathway, was studied in experimental autoimmune neuritis. In the absence of TLR2, TLR4, myeloid differentiation response gene 88, or TRIF, the clinical course was significantly attenuated compared to wild-type mice. This could be attributed to impaired NF-κB activation, as shown by the absence of nuclear translocation of RelA with a decreased expression of IL-6, IL-12p40, and IL-17A. Remarkably, P0106-125-immunized TLR20/0 mice exhibited a delayed recovery as compared to TLR40/0 mice, which was because of an impaired T helper cell 2 polarization. Immunized TLR20/0 mice were unable to induce OX40 and OX40L by matrix metalloproteinase-2 on splenic dendritic cells. Subsequently, M2 polarization was impaired and macrophages were unable to sufficiently induce T regulatory cells (Tregs). Thus, in the recovery phase, Tregs were significantly increased in TLR40/0 mice as compared to wild-type mice, whereas Tregs in immunized TLR20/0 mice were only slightly increased. Our data highlight the relevance of innate immunity and, especially, the tight interaction between the innate and the adaptive immune system, which should be considered for therapeutic approaches of autoimmune diseases.

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses.

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cell responses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these 'endogenous' NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-TNP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-TNP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.

IL-10, IL-4, and STAT6 promote an M2 milieu required for termination of P0(106-125)-induced murine experimental autoimmune neuritis.

The role of the type 2 helper T cell (Th2)-polarizing cytokines IL-4 and IL-10 has not yet been studied in P0106-125-induced murine experimental autoimmune neuritis (EAN). We, therefore, addressed the functional relevance of these cytokines and signaling via the IL-4-associated transcription factor STAT6. The clinical course of P0106-125-induced EAN in mice deficient for IL-10(0/0), IL-4(0/0), or STAT6(0/0) was significantly aggravated compared with that of wild-type control mice. In addition, treatment of P0106-125-immunized C57BL/6 mice at the onset of clinical symptoms with a monoclonal IL-10 neutralizing antibody aggravated symptoms and prolonged disease to a similar degree as in IL-10(0/0) mice. This exacerbated course was attributed to a more prominent Th1 immune response associated with a persistent M1 milieu in the sciatic nerve and in the regional and systemic lymphatic system. These data suggest a Th2-polarized milieu being required to prevent axonal damage of the sciatic nerve and to terminate the P0106-125-specific immune response in EAN. Beyond the already known role of macrophages as pathogenic effector cells in EAN, these data suggest that M2-differentiated macrophages do not damage and may even protect neural tissues in EAN. Thus, these data highlight the pathogenetic relevance of the macrophage polarization status in EAN. Therapeutic modulation of immune responses from an M1 toward an M2 milieu may be a promising novel strategy in peripheral nervous system neuritis.

The N-terminal domain of NLRC5 confers transcriptional activity for MHC class I and II gene expression.

Ag presentation to CD4(+) and CD8(+) T cells depends on MHC class II and MHC class I molecules, respectively. One important regulatory factor of this process is the transcriptional regulation of MHC gene expression. It is well established that MHC class II transcription relies on the NLR protein CIITA. Recently, another NLR protein, NLRC5, was shown to drive MHC class I expression. The molecular mechanisms of the function of NLRC5 however remain largely elusive. In this study, we present a detailed functional study of the domains of NLRC5 revealing that the N-terminal domain of human NLRC5 has intrinsic transcriptional activity. Domain swapping experiments between NLRC5 and CIITA showed that this domain contributes to MHC class I and MHC class II gene expression with a bias for activation of MHC class I promoters. Delivery of this construct by adeno-associated viral vectors upregulated MHC class I and MHC class II expression in human cells and enhanced lysis of melanoma cells by CD8(+) cytotoxic T cells in vitro. Taken together, this work provides novel insight into the function of NLRC5 and CIITA in MHC gene regulation.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Costimulatory molecule CD40 is essential for myelin protein 0 peptide 106-125-induced experimental autoimmune neuritis in mice.

Myelin protein 0 peptide 106-125-induced murine experimental autoimmune neuritis (EAN) is a CD4-positive T cell-mediated monophasic axonal inflammatory neuropathy; interferon-γ is the key proinflammatory mediator. Experimental autoimmune neuritis is well suited for elucidating pathogenetic mechanisms underlying human acute axonal Guillain-Barré syndrome. Here, the functional role of the costimulatory molecule CD40 was defined by characterization of EAN in CD40-deficient mice. In contrast to immunized C57BL/6 mice, CD40-deficient mice were resistant to EAN owing to impaired priming of CD4-positive T-effector cells. To determine whether CD40 is a suitable candidate for the treatment of EAN, we administered monoclonal anti-CD40 antibody either before immunization or upon onset of neurologic signs. Prophylactic anti-CD40 treatment completely abolished CD4-positive T-cell priming. Therapeutic application of anti-CD40 prevented full activation of CD4-positive T cells that were in the process of priming and suppressed production of interferon-γ in peripheral lymph nodes, spleen, and serum, and of interleukin-6, interleukin-12p40, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, which are associated with activation of the nuclear factor-κB signaling pathway. This resulted in enhanced recovery by early generation of CD25-positive, Foxp3-positive, CD4-positive regulatory T cells. Thus, these experiments highlight the crucial role of CD40 as an important costimulatory molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis.

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.