GENETIC ABLATION OF THE C-TYPE LECTIN RECEPTOR CLEC2D INCREASES PERITONITIS MORTALITY, INFLAMMATION AND PHYSIOLOGY WITHOUT DIMINISHING ORGAN INJURY.

BACKGROUND: Sepsis accounts for substantial morbidity and mortality motivating investigators to continue the search for pathways and molecules driving the pathogenesis of the disease. The current study examined if the novel C-type Lectin Receptor (CLR), Clec2d, plays a significant role in the pathogenesis of sepsis. METHODS: Clec2d knockout (KO) mice were fully backcrossed onto the C57\BL6 background. Acute endotoxemia was induced with an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Sepsis was induced in two different models, Cecal Ligation and Puncture (CLP) and Pseudomonas aeruginosa pneumonia. Both models were treated with antibiotics and fluid resuscitation. In the sepsis models, physiologic and hematologic measurements were measured at 24 hours by collecting a small sample of peripheral blood. Mortality was followed for 14 days. RESULTS: A total of 197 mice were studied, 58 wild type (WT) and 54 knock-out (KO) in the LPS model; 27 wild type and 21 KO mice in the CLP model; and 22 WT and 15 KO mice in the pneumonia model. Clec2d KO mice had greater mortality in the LPS and CLP studies but not the pneumonia model. There were significant differences in multiple parameters determined 24 hours post sepsis between mice who would subsequently died and those lived. Consistent with previous reports in the CLP model, higher concentrations of IL-6, increased numbers of peripheral blood lymphocytes and greater renal injury were found in the dying mice. In contrast, in the pneumonia model IL-6 was higher in the surviving mice, however, the IL-6 levels in the pneumonia model (0.6 ± 0.3 ng/ml mean ± SEM) were less than 2% of the IL-6 levels of mice that died in the CLP model (41 ± 9 ng/ml, mean ± SEM). There were no differences in the lymphocyte count or renal injury between living and dying mice in the pneumonia model. In both sepsis models dying mice had lower heart rates, respiratory rates, and body temperatures. These values were also lower in the KO mice compared to the WT in CLP, but the breath rate and body temperature were increased in the KO pneumonia mice. CONCLUSION: The C-type lectin receptor Clec2d plays a complicated role in the pathogenesis of sepsis which varies with source of infection as demonstrated in the models used to study the disease. These data highlight the heterogeneity of the responses to sepsis and provide further evidence that a single common pathway driving sepsis organ injury and death likely does not exist.

Tumor immune evasion through loss of MHC class-I antigen presentation.

CD8 T cells recognize cancers when they detect antigenic peptides presented on a tumor's surface MHC-I molecules. Since MHC-I antigen presentation is not essential for cell growth or survival, many cancers inactivate this pathway, and thereby escape control by CD8 T cells. Such immune evasion allows cancers to progress and also become resistant to CD8 T- cell-based immunotherapies, such as checkpoint blockade. Here, we review recent findings about the various different mechanisms that cancers use to impair antigen presentation, the consequence of such changes, and, in some cases, the potential to reverse these defects.

Pathways of MHC I cross-presentation of exogenous antigens.

Phagocytes, particularly dendritic cells (DCs), generate peptide-major histocompatibility complex (MHC) I complexes from antigens they have collected from cells in tissues and report this information to CD8 T cells in a process called cross-presentation. This process allows CD8 T cells to detect, respond and eliminate abnormal cells, such as cancers or cells infected with viruses or intracellular microbes. In some settings, cross-presentation can help tolerize CD8 T cells to self-antigens. One of the principal ways that DCs acquire tissue antigens is by ingesting this material through phagocytosis. The resulting phagosomes are key hubs in the cross-presentation (XPT) process and in fact experimentally conferring the ability to phagocytize antigens can be sufficient to allow non-professional antigen presenting cells (APCs) to cross-present. Once in phagosomes, exogenous antigens can be cross-presented (XPTed) through three distinct pathways. There is a vacuolar pathway in which peptides are generated and then bind to MHC I molecules within the confines of the vacuole. Ingested exogenous antigens can also be exported from phagosomes to the cytosol upon vesicular rupture and/or possibly transport. Once in the cytosol, the antigen is degraded by the proteasome and the resulting oligopeptides can be transported to MHC I molecule in the endoplasmic reticulum (ER) (a phagosome-to-cytosol (P2C) pathway) or in phagosomes (a phagosome-to-cytosol-to-phagosome (P2C2P) pathway). Here we review how phagosomes acquire the necessary molecular components that support these three mechanisms and the contribution of these pathways. We describe what is known as well as the gaps in our understanding of these processes.

Tetraspanin-5-mediated MHC class I clustering is required for optimal CD8 T cell activation.

MHC molecules are not randomly distributed on the plasma membrane but instead are present in discrete nanoclusters. The mechanisms that control formation of MHC I nanoclusters and the importance of such structures are incompletely understood. Here, we report a molecular association between tetraspanin-5 (Tspan5) and MHC I molecules that started in the endoplasmic reticulum and was maintained on the plasma membrane. This association was observed both in mouse dendritic cells and in human cancer cell lines. Loss of Tspan5 reduced the size of MHC I clusters without affecting MHC I peptide loading, delivery of complexes to the plasma membrane, or overall surface MHC I levels. Functionally, CD8 T cell responses to antigen presented by Tspan5-deficient dendritic cells were impaired but were restored by antibody-induced reclustering of MHC I molecules. In contrast, Tspan5 did not associate with two other plasma membrane proteins, Flotillin1 and CD55, with or the endoplasmic reticulum proteins Tapasin and TAP. Thus, our findings identify a mechanism underlying the clustering of MHC I molecules that is important for optimal T cell responses.

Cancer Immune Evasion Through Loss of MHC Class I Antigen Presentation.

Major histocompatibility class I (MHC I) molecules bind peptides derived from a cell's expressed genes and then transport and display this antigenic information on the cell surface. This allows CD8 T cells to identify pathological cells that are synthesizing abnormal proteins, such as cancers that are expressing mutated proteins. In order for many cancers to arise and progress, they need to evolve mechanisms to avoid elimination by CD8 T cells. MHC I molecules are not essential for cell survival and therefore one mechanism by which cancers can evade immune control is by losing MHC I antigen presentation machinery (APM). Not only will this impair the ability of natural immune responses to control cancers, but also frustrate immunotherapies that work by re-invigorating anti-tumor CD8 T cells, such as checkpoint blockade. Here we review the evidence that loss of MHC I antigen presentation is a frequent occurrence in many cancers. We discuss new insights into some common underlying mechanisms through which some cancers inactivate the MHC I pathway and consider some possible strategies to overcome this limitation in ways that could restore immune control of tumors and improve immunotherapy.

How a tailor achieves the perfect fit.

Most antigenic peptides that bind stably to a major histocompatibility complex (MHC) I molecule for display to the immune system are approximately the same length, thanks in part to the expert trimming done by endoplasmic reticulum aminopeptidases (ERAPs), the final peptidases in the antigen-presentation pathway. An open question is whether ERAPs edit peptides to this optimal length while they are bound to MHC I molecules (using the latter as a pattern of sorts) or by free hand. Mavridis et al. present multiple lines of evidence that this trimming cannot readily occur on MHC I molecules, but rather only in solution, suggesting that ERAPs work alone to tailor the perfect fit for the immunopeptidome.

Cross-presentation of exogenous antigens on MHC I molecules.

In order to get recognized by CD8 T cells, most cells present peptides from endogenously expressed self or foreign proteins on MHC class I molecules. However, specialized antigen-presenting cells, such as DCs and macrophages, can present exogenous antigen on MHC-I in a process called cross-presentation. This pathway plays key roles in antimicrobial and antitumor immunity, and also immune tolerance. Recent advances have broadened our understanding of the underlying mechanisms of cross-presentation. Here, we review some of these recent advances, including the distinct pathways that result in the cross-priming of CD8 T cells and the source of the class I molecules presenting exogenous peptides.

The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments.

For CD8 T lymphocytes to mount responses to cancer and virally-infected cells, dendritic cells must capture antigens present in tissues and display them as peptides bound to MHC-I molecules. This is most often accomplished through a pathway called antigen cross-presentation (XPT). Here, we report that the vesicular trafficking protein Rab39a is needed for optimal cross-presentation by dendritic cells in vitro and cross-priming of CD8 T cells in vivo. Without Rab39a, MHC-I presentation of intraphagosomal peptides is inhibited, indicating that Rab39a converts phagosomes into peptide-loading compartments. In this process, Rab39a promotes the delivery of MHC-I molecules from the endoplasmic reticulum (ER) to phagosomes, and increases the levels of peptide-empty MHC-I conformers that can be loaded with peptide in this compartment. Rab39a also increases the levels of Sec22b and NOX2, previously recognized to participate in cross-presentation, on phagosomes, thereby filling in a missing link into how phagosomes mature into cross-presenting vesicles.

Immune Sensing of Cell Death through Recognition of Histone Sequences by C-Type Lectin-Receptor-2d Causes Inflammation and Tissue Injury.

The immune system monitors the health of cells and is stimulated by necrosis. Here we examined the receptors and ligands driving this response. In a targeted screen of C-type lectin receptors, a Clec2d reporter responded to lysates from necrotic cells. Biochemical purification identified histones, both free and bound to nucleosomes or neutrophil extracellular traps, as Clec2d ligands. Clec2d recognized poly-basic sequences in histone tails and this recognition was sensitive to post-translational modifications of these sequences. As compared with WT mice, Clec2d-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model. In macrophages, Clec2d localized to the plasma membrane and endosomes. Histone binding to Clec2d did not stimulate kinase activation or cytokine production. Rather, histone-bound DNA stimulated endosomal Tlr9-dependent responses in a Clec2d-dependent manner. Thus, Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage.

Frequent Loss of IRF2 in Cancers Leads to Immune Evasion through Decreased MHC Class I Antigen Presentation and Increased PD-L1 Expression.

To arise and progress, cancers need to evade immune elimination. Consequently, progressing tumors are often MHC class I (MHC-I) low and express immune inhibitory molecules, such as PD-L1, which allows them to avoid the main antitumor host defense, CD8+ T cells. The molecular mechanisms that led to these alterations were incompletely understood. In this study, we identify loss of the transcription factor IRF2 as a frequent underlying mechanism that leads to a tumor immune evasion phenotype in both humans and mice. We identified IRF2 in a CRISPR-based forward genetic screen for genes that controlled MHC-I Ag presentation in HeLa cells. We then found that many primary human cancers, including lung, colon, breast, prostate, and others, frequently downregulated IRF2. Although IRF2 is generally known as a transcriptional repressor, we found that it was a transcriptional activator of many key components of the MHC-I pathway, including immunoproteasomes, TAP, and ERAP1, whose transcriptional control was previously poorly understood. Upon loss of IRF2, cytosol-to-endoplasmic reticulum peptide transport and N-terminal peptide trimming become rate limiting for Ag presentation. In addition, we found that IRF2 is a repressor of PD-L1. Thus, by downregulating a single nonessential gene, tumors become harder to see (reduced Ag presentation), more inhibitory (increased checkpoint inhibitor), and less susceptible to being killed by CD8+ T cells. Importantly, we found that the loss of Ag presentation caused by IRF2 downregulation could be reversed by IFN-stimulated induction of the transcription factor IRF1. The implication of these findings for tumor progression and immunotherapy are discussed.

The Biology and Underlying Mechanisms of Cross-Presentation of Exogenous Antigens on MHC-I Molecules.

To monitor the health of cells, the immune system tasks antigen-presenting cells with gathering antigens from other cells and bringing them to CD8 T cells in the form of peptides bound to MHC-I molecules. Most cells would be unable to perform this function because they use their MHC-I molecules to exclusively present peptides derived from the cell's own proteins. However, the immune system evolved mechanisms for dendritic cells and some other phagocytes to sample and present antigens from the extracellular milieu on MHC-I through a process called cross-presentation. How this important task is accomplished, its role in health and disease, and its potential for exploitation are the subject of this review.

Frontline Science: Multiple cathepsins promote inflammasome-independent, particle-induced cell death during NLRP3-dependent IL-1ß activation.

Sterile particles cause several chronic, inflammatory diseases, characterized by repeating cycles of particle phagocytosis and inflammatory cell death. Recent studies have proposed that these processes are driven by the NLRP3 inflammasome, a platform activated by phagocytosed particles, which controls both caspase-1-dependent cell death (pyroptosis) and mature IL-1ß secretion. After phagocytosis, particles can disrupt lysosomes, and inhibitor studies have suggested that the resulting release of a lysosomal protease-cathepsin B-into the cytosol somehow activates NLRP3. However, using primary murine macrophages, we found that particle-induced cell death occurs independent of NLRP3/caspase-1 and depends instead on multiple, redundant cathepsins. In contrast, nigericin, a soluble activator of NLRP3 inflammasomes, induced cell death that was dependent on the NLRP3. Interestingly, nigericin-induced cell death depended partly on a single cathepsin, cathepsin X. By inhibiting or silencing multiple cathepsins in macrophages, several key proinflammatory events induced by sterile particles are blocked, including cell death, pro-IL-1ß production, and IL-1ß secretion. These data suggest that cathepsins might be potential therapeutic targets in particulate-mediated inflammatory disease. In support of this concept, we find that a broad-spectrum cathepsin inhibitor can suppress particle-induced IL-1-dependent peritonitis.

Present Yourself! By MHC Class I and MHC Class II Molecules.

Since the discovery of MHC molecules, it has taken 40 years to arrive at a coherent picture of how MHC class I and MHC class II molecules really work. This is a story of the proteases and MHC-like chaperones that support the MHC class I and II molecules in presenting peptides to the immune system. We now understand that the MHC system shapes both the repertoire of presented peptides and the subsequent T cell response, with important implications ranging from transplant rejection to tumor immunotherapies. Here we present an illustrated review of the ins and outs of MHC class I and MHC class II antigen presentation.

Specialized proteasome subunits have an essential role in the thymic selection of CD8(+) T cells.

The cells that stimulate positive selection express specialized proteasome ß-subunits different from those expressed by all other cells, including those involved in negative selection. Mice that lack all four specialized proteasome ß-subunits, and therefore express only constitutive proteasomes in all cells, had a profound defect in the generation of CD8(+) T cells. While a defect in positive selection would reflect an inability to generate the appropriate positively selecting peptides, a block at negative selection would point to the potential need to switch peptides between positive selection and negative selection to avoid the two processes' often cancelling each other out. We found that the block in T cell development occurred around the checkpoints of positive selection and, unexpectedly, negative selection as well.

The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8+ T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi.

The ß1i, ß2i and ß5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected ß1i, ß2i and ß5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.

Multiple Cathepsins Promote Pro-IL-1ß Synthesis and NLRP3-Mediated IL-1ß Activation.

Sterile particles induce robust inflammatory responses that underlie the pathogenesis of diseases like silicosis, gout, and atherosclerosis. A key cytokine mediating this response is IL-1ß. The generation of bioactive IL-1ß by sterile particles is mediated by the NOD-like receptor containing a pyrin domain 3 (NLRP3) inflammasome, although exactly how this occurs is incompletely resolved. Prior studies have found that the cathepsin B inhibitor, Ca074Me, suppresses this response, supporting a model whereby ingested particles disrupt lysosomes and release cathepsin B into the cytosol, somehow activating NLRP3. However, reports that cathepsin B-deficient macrophages have no defect in particle-induced IL-1ß generation have questioned cathepsin B's involvement. In this study, we examine the hypothesis that multiple redundant cathepsins (not just cathepsin B) mediate this process by evaluating IL-1ß generation in murine macrophages, singly or multiply deficient in cathepsins B, L, C, S and X. Using an activity-based probe, we measure specific cathepsin activity in living cells, documenting compensatory changes in cathepsin-deficient cells, and Ca074Me's dose-dependent cathepsin inhibition profile is analyzed in parallel with its suppression of particle-induced IL-1ß secretion. Also, we evaluate endogenous cathepsin inhibitors cystatins C and B. Surprisingly, we find that multiple redundant cathepsins, inhibited by Ca074Me and cystatins, promote pro-IL-1ß synthesis, and to our knowledge, we provide the first evidence that cathepsin X plays a nonredundant role in nonparticulate NLRP3 activation. Finally, we find cathepsin inhibitors selectively block particle-induced NLRP3 activation, independently of suppressing pro-IL-1ß synthesis. Altogether, we demonstrate that both small molecule and endogenous cathepsin inhibitors suppress particle-induced IL-1ß secretion, implicating roles for multiple cathepsins in both pro-IL-1ß synthesis and NLRP3 activation.

The xanthine oxidase inhibitor Febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung.

Necrotic cell death in vivo induces a robust neutrophilic inflammatory response and the resulting inflammation can cause further tissue damage and disease. Dying cells induce this inflammation by releasing pro-inflammatory intracellular components, one of which is uric acid. Cells contain high levels of intracellular uric acid, which is produced when purines are oxidized by the enzyme xanthine oxidase. Here we test whether a non-nucleoside xanthine oxidase inhibitor, Febuxostat (FBX), can reduce intracellular uric acid levels and inhibit cell death-induced inflammation in two different murine tissue injury models; acid-induced acute lung injury and acetaminophen liver injury. Infiltration of inflammatory cells induced by acid injection into lungs or peritoneal administration of acetaminophen was evaluated by quantification with flow cytometry and tissue myeloperoxidase activity in the presence or absence of FBX treatment. Uric acid levels in serum and tissue were measured before giving the stimuli and during inflammation. The impact of FBX treatment on the peritoneal inflammation caused by the microbial stimulus, zymosan, was also analyzed to see whether FBX had a broad anti-inflammatory effect. We found that FBX reduced uric acid levels in acid-injured lung tissue and inhibited acute pulmonary inflammation triggered by lung injury. Similarly, FBX reduced uric acid levels in the liver and inhibited inflammation in response to acetaminophen-induced hepatic injury. In contrast, FBX did not reduce inflammation to zymosan, and therefore is not acting as a general anti-inflammatory agent. These results point to the potential of using agents like FBX to treat cell death-induced inflammation.

Evaluation of the contribution of multiple DAMPs and DAMP receptors in cell death-induced sterile inflammatory responses.

When cells die by necrosis in vivo they stimulate an inflammatory response. It is thought that this response is triggered when the injured cells expose proinflammatory molecules, collectively referred to as damage associated molecular patterns (DAMPs), which are recognized by cells or soluble molecules of the innate or adaptive immune system. Several putative DAMPs and/or their receptors have been identified, but whether and how much they participate in responses in vivo is incompletely understood, and they have not previously been compared side-by-side in the same models. This study focuses on evaluating the contribution of multiple mechanisms that have been proposed to or potentially could participate in cell death-induced inflammation: The third component of complement (C3), ATP (and its receptor P2X7), antibodies, the C-type lectin receptor Mincle (Clec4e), and protease-activated receptor 2 (PAR2). We investigate the role of these factors in cell death-induced inflammation to dead cells in the peritoneum and acetaminophen-induced liver damage. We find that mice deficient in antibody, C3 or PAR2 have impaired inflammatory responses to dying cells. In contrast there was no reduction in inflammation to cell death in the peritoneum or liver of mice that genetically lack Mincle, the P2X7 receptor or that were treated with apyrase to deplete ATP. These results indicate that antibody, complement and PAR2 contribute to cell death-induced inflammation but that Mincle and ATP- P2X7 receptor are not required for this response in at least 2 different in vivo models.