Intragranuloma Accumulation and Inflammatory Differentiation of Neutrophils Underlie Mycobacterial ESX-1-Dependent Immunopathology.

The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6C+MHCII+ monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6C+MHCII+ monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. IMPORTANCE The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.

IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDC1 into the cDC2 lineage.

Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1. In the absence of Irf8, committed cDC1 acquired the transcriptional, functional, and chromatin accessibility properties of cDC2. This conversion was independent of Irf4 and was associated with the decreased accessibility of putative IRF8, Batf3, and composite AP-1-IRF (AICE)-binding elements, together with increased accessibility of cDC2-associated transcription-factor-binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2-like cells.

Intestinal cDC1 drive cross-tolerance to epithelial-derived antigen via induction of FoxP3+CD8+ Tregs.

Although CD8+ T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)-derived antigen involves the generation and suppressive function of FoxP3+CD8+ T cells. FoxP3+CD8+ Treg generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction. Mechanistically, intestinal cDC1-derived PD-L1, TGFß, and retinoic acid contributed to the generation of gut-tropic CCR9+CD103+FoxP3+CD8+ Tregs Last, CD103-deficient CD8+ T cells lacked tolerogenic activity in vivo, indicating a role for CD103 in FoxP3+CD8+ Treg function. Our results describe a role for FoxP3+CD8+ Tregs in cross-tolerance in the intestine for which development requires intestinal cDC1.

Complete Topological Mapping of a Cellular Protein Interactome Reveals Bow-Tie Motifs as Ubiquitous Connectors of Protein Complexes.

The network topology of a protein interactome is shaped by the function of each protein, making it a resource of functional knowledge in tissues and in single cells. Today, this resource is underused, as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell, thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins, each forming topological motifs called "bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties, display multifunctional capabilities, are enriched for essential proteins, and are widely expressed in other cells and tissues. Collectively, the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such, these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates.

Prevention of colitis-associated cancer by selective targeting of immunoproteasome subunit LMP7.

Chronic inflammation is a well-known risk factor in development of intestinal tumorigenesis, although the exact mechanisms underlying development of colitis-associated cancer (CAC) still remain obscure. The activity and function of immunoproteasome has been extensively analyzed in the context of inflammation and infectious diseases. Here, we show that the proteasomal immunosubunit LMP7 plays an essential role in development of CAC. Mice devoid of LMP7 were resistant to chronic inflammation and formation of neoplasia, and developed virtually no tumors after AOM/DSS treatment. Our data reveal that LMP7 deficiency resulted in reduced expression of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3 as well as adhesion molecule VCAM-1. As a consequence, an impaired recruitment and activity of tumor-infiltrating leukocytes resulting in decreased secretion of cytokines IL-6 and TNF-α was observed. Further, the deletion or pharmacological inhibition of LMP7 and consequent blockade of NF-κB abrogated the production of IL-17A, which possesses a strong carcinogenic activity in the gut. Moreover, in vivo administration of the selective LMP7 inhibitor ONX-0914 led to a marked reduction of tumor numbers in wild-type (WT) mice. Collectively, we identified the immunoproteasome as a crucial mediator of inflammation-driven neoplasia highlighting a novel potential therapeutic approach to limit colonic tumorigenesis.

IRF8 Transcription Factor Controls Survival and Function of Terminally Differentiated Conventional and Plasmacytoid Dendritic Cells, Respectively.

Interferon regulatory factor-8 (IRF8) has been proposed to be essential for development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) and remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells are designated "terminal selectors." Using BM chimeras, conditional Irf8(fl/fl) mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene-expression profile leading to increased T cell stimulatory function but decreased type 1 interferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s, and pDCs.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis.

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αß(+) and CD4(+)CD8αα(+) T cells; the latter requiring ß8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors.

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

The proteasome system in infection: impact of ß5 and LMP7 on composition, maturation and quantity of active proteasome complexes.

Proteasomes are the major enzyme complexes for non-lysosomal protein degradation in eukaryotic cells. Mammals express two sets of catalytic subunits: the constitutive subunits ß1, ß2 and ß5 and the immunosubunits LMP2 (ß1i), MECL-1 (ß2i) and LMP7 (ß5i). The LMP7-propeptide (proLMP7) is required for optimal maturation of LMP2/MECL-1-containing precursors to mature immunoproteasomes, but can also mediate efficient integration into mixed proteasomes containing ß1 and ß2. In contrast, the ß5-propeptide (proß5) has been suggested to promote preferential integration into ß1/ß2-containing precursors, consequently favouring the formation of constitutive proteasomes. Here, we show that proß5 predominantly promotes integration into LMP2/MECL-1-containing precursors in IFNγ-stimulated, LMP7-deficient cells and infected LMP7-deficient mice. This demonstrates that proß5 does not direct preferential integration into ß1/ß2-containing precursors, but instead promotes the formation of mixed LMP2/MECL-1/ß5 proteasomes under inflammatory conditions. Moreover, the propeptides substantially differ in their capacity to promote proteasome maturation, with proLMP7 showing a significantly higher chaperone activity as compared to proß5. Increased efficiency of proteasome maturation mediated by proLMP7 is required for optimal MHC class I cell surface expression and is equally important as the catalytic activity of immunoproteasomes. Intriguingly, induction of LMP7 by infection not only results in rapid exchange of constitutive by immunosubunits, as previously suggested, but also increases the total proteasome abundance within the infected tissue. Hence our data identify a novel LMP7-dependend mechanism to enhance the activity of the proteasome system in infection, which is based on the high chaperone activity of proLMP7 and relies on accelerated maturation of active proteasome complexes.

c-Rel promotes type 1 and type 17 immune responses during Leishmania major infection.

Recent studies demonstrated the crucial role of c-Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell-mediated autoimmune inflammation. We thus wondered whether these opposite functions of c-Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T-cell subsets on disease outcome. Here we show that c-Rel-deficient (rel(-/-) ) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN-γ and IL-17 production in response to L. major antigens was severely impaired in rel(-/-) mice. Reconstitution of Rag1(-/-) T-cell deficient mice with rel(-/-) CD4(+) T cells followed by L. major infection demonstrated that c-Rel-deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4(+) cells in vitro was normal. Notably, a selective defect in IL-12 and IL-23 production was observed in rel(-/-) DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c-Rel in myeloid cells is essential for clearance of L. major and that this c-Rel-mediated effect is dominant over the lack of Tregs.

Targeting the proteasome: partial inhibition of the proteasome by bortezomib or deletion of the immunosubunit LMP7 attenuates experimental colitis.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD), comprising Crohn s disease and ulcerative colitis, is characterised by chronic relapsing inflammation of the gut. Increased proteasome activity, associated with the expression of immunoproteasomes, was found to enhance proinflammatory signalling and thus promotes inflammation in patients with IBD. The aim of this study was to explore whether modulation of the proteasomal activity is a suitable therapeutic approach to limit inflammation in colitis. METHODS: This concept was assessed in two different experimental set-ups. Development of dextran sodium sulfate (DSS)-induced colitis was tested (1) in lmp7(-/-) mice lacking the immunoproteasome subunit LMP7 and (2) in wild-type (WT) mice treated with the proteasome inhibitor bortezomib. RESULTS: Compared with WT mice, lmp7(-/-) mice develop significantly attenuated colitis due to reduced nuclear factor-kappaB (NF-kappaB) signalling in the absence of LMP7. Further, treatment with bortezomib revealed dose-dependent amelioration of DSS-induced inflammation. In both approaches modulation of the proteasome activity limited the secretion of proinflammatory cytokines and chemokines. Consequently, infiltration of the colon by neutrophils and expansion of inflammatory T helper 1 (Th1) and Th17 T cells was diminished and thus prevented excessive tissue damage. CONCLUSIONS: It was demonstrated that modulation of the proteasome activity is effective in attenuating experimental colitis. The results reveal that reduction of the proteasome activity either by partial inhibition with bortezomib or by specifically targeting the immunoproteasome subunit LMP7 is a suitable treatment of intestinal inflammation.

c-Rel is crucial for the induction of Foxp3(+) regulatory CD4(+) T cells but not T(H)17 cells.

The NF-kappaB/Rel family member c-Rel was described to be required for the development of T(H)1 responses. However, the role of c-Rel in the differentiation of T(H)17 and regulatory CD4(+)Foxp3(+) T cells (Treg) remains obscure. Here, we show that in the absence of c-Rel, in vitro differentiation of pro-inflammatory T(H)17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c-Rel-deficient CD4(+) T cells was severely hampered and correlated to reduced numbers of Foxp3(+) T cells in vivo. Mechanistically, in vitro conversion of naive CD4(+) T cells into iTreg was crucially dependent on c-Rel-mediated synthesis of endogenous IL-2. The addition of exogenous IL-2 was sufficient to rescue the development of c-Rel-deficient iTreg. Thus, c-Rel is essential for the development of Foxp3(+) Treg but not for T(H)17 cells via regulating the production of IL-2.

Comparative expression analysis and characterization of 20S proteasomes in human intestinal tissues: The proteasome pattern as diagnostic tool for IBD patients.

BACKGROUND: The diagnostic differentiation between Crohn's disease (CD) and ulcerative colitis (UC) is sometimes difficult. To date, there are no serological markers that are specific and sensitive enough to differentiate between these 2 diseases. Early and safe prediction of the inflammatory bowel disease (IBD) type is of great importance for the specific treatment of IBD patients. We thus analyzed and compared the expression of catalytic proteasome subunits in the gut of mice and in the normal and inflamed intestines of CD and UC patients and assessed whether the subunit pattern is suitable for diagnostic differentiation. METHODS: The 20S proteasomes were isolated from surgical tissue specimens derived from terminal ileum and colon of IBD patients and controls. Spots of 20S proteasomes separated by 2D electrophoresis were analyzed by mass spectrometry. Quick detection of catalytic beta2, beta2i, and beta5i subunits was performed by incubating proteasomes with a biotinylated inhibitor (AdaK(Bio)Ahx3L3VS) and subsequently by streptavidin-horseradish peroxide. RESULTS: 20S proteasomes were isolated from the human liver, colon, and terminal ileum. Low expression of the immunosubunits beta1i and beta2i was found in the liver and colon but high amounts in the small intestine. In colon and liver beta5i was found to be associated with the constitutive beta1, beta2 subunits, indicating the existence of mixed proteasomes. Further, inflammation in CD but not UC patients induced massive upregulation of beta1i and beta2i in the colon and terminal ileum, indicating the importance of this protein complex as a disease marker. CONCLUSIONS: We here show that CD and UC patients display a characteristic pattern of proteasome subunit composition which can be used as diagnostic tool to differentiate between CD and UC.

Proteasome-mediated degradation of IkappaBalpha and processing of p105 in Crohn disease and ulcerative colitis.

Enhanced NF-kappaB activity is involved in the pathology of both forms of inflammatory bowel disease (IBD), Crohn disease (CD) and ulcerative colitis (UC). Here we analyzed the mechanism of proteasome-mediated NF-kappaB activation in CD and UC. Our studies demonstrate that the subunit composition and the proteolytic function of proteasomes differ between UC and CD. High expression of the immunoproteasome subunits beta1i and beta2i is characteristic of the inflamed mucosa of CD. In line with this, we found enhanced processing of NF-kappaB precursor p105 and degradation of inhibitor of NF-kappaB, IkappaBalpha, by immunoproteasomes isolated from the mucosa of CD patients. In comparison with healthy controls and CD patients, UC patients exhibited an intermediate phenotype regarding the proteasome-mediated processing/degradation of NF-kappaB components. Finally, increased expression of the NF-kappaB family member c-Rel in the inflamed mucosa of CD patients suggests that p50/c-Rel is important for IFN-gamma-mediated induction of immunoproteasomes via IL-12-driven Th1 responses. These findings suggest that distinct proteasome subunits influence the intensity of NF-kappaB-mediated inflammation in IBD patients.

Immunoproteasomes are essential for clearance of Listeria monocytogenes in nonlymphoid tissues but not for induction of bacteria-specific CD8+ T cells.

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8(+) T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8(+) T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8(+) T cell epitopes. I-proteasome-deficient mice (beta5i(-/-) mice) exhibited normal frequencies of L. monocytogenes-specific CD8(+) T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8(+) T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.

Quantification of photosynthetic gene expression in maize C(3) and C(4) tissues by real-time PCR.

Carbon assimilation in maize follows the C(4) mechanism. This requires the tissue-specific and light-induced expression of a set of different genes involved in CO(2) fixation as well as adaptations in the leaf anatomy including a reduced distance between vascular bundles compared to C(3) plants. However, several maize tissues exist with larger bundle distances and there is significant evidence that CO(2) fixation follows the C(3) mechanism in these tissues. We isolated maize C(3) and C(4) tissues and quantified the accumulation of mRNAs encoding PEPC, ME, the small subunit of Rubisco, and PPDK. For this, primer systems for the specific and sensitive detection by real-time PCR were established. The observed patterns show the expected distribution for foliar leaf tissues. Also in total husk leaves, all transcripts under investigation were detected, albeit at a lower level. When mesophyll cells which are located distant from bundles were isolated from husk leaves, only accumulation of RbcS was observed. Comparing the expression of two genes encoding for isoenzymes of the small subunit of RbcS in the different tissues differential patterns of relative transcript abundance were observed. Transcripts for the DOF1 transcription factor involved in the activation of photosynthetic genes in maize were found in leaf tissues performing both C(4) and C(3) photosynthesis with highest accumulation levels in C(4) mesophyll cells, whereas the homologous DOF2 gene was not expressed in any of the investigated samples. The results provide novel insights into the regulation of C(3) and C(4) carbon fixation pathways in maize.