The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity.

The molecules and pathways that fine-tune innate inflammatory responses mediated by Toll-like receptor 7 (TLR7) remain to be fully elucidated. Using an unbiased genome-scale screen with short hairpin RNA (shRNA), we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4(-/-) mice were hyporesponsive to TLR7 agonists and failed to produce type I interferons due to impaired phosphorylation of the transcription factor STAT1 by the mitogen-activated protein kinase p38 and decreased recruitment of the adaptor MyD88 to TLR7. TREML4 deficiency reduced the production of inflammatory cytokines and autoantibodies in MRL/lpr mice, which are prone to systemic lupus erythematosus (SLE), and inhibited the antiviral immune response to influenza virus. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity.

The scavenger receptor SCARF1 mediates the clearance of apoptotic cells and prevents autoimmunity.

The clearance of apoptotic cells is critical for the control of tissue homeostasis; however, the full range of receptors on phagocytes responsible for the recognition of apoptotic cells remains to be identified. Here we found that dendritic cells (DCs), macrophages and endothelial cells used the scavenger receptor SCARF1 to recognize and engulf apoptotic cells via the complement component C1q. Loss of SCARF1 impaired the uptake of apoptotic cells. Consequently, in SCARF1-deficient mice, dying cells accumulated in tissues, which led to a lupus-like disease, with the spontaneous generation of autoantibodies to DNA-containing antigens, activation of cells of the immune system, dermatitis and nephritis. The discovery of such interactions of SCARF1 with C1q and apoptotic cells provides insight into the molecular mechanisms involved in the maintenance of tolerance and prevention of autoimmune disease.

Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation.

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.

SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

Deficiency in the clearance of cellular debris is a major pathogenic factor in the emergence of autoimmune diseases. We previously demonstrated that mice deficient for scavenger receptor class F member 1 (SCARF1) develop a lupus-like autoimmune disease with symptoms similar to human systemic lupus erythematosus (SLE), including a pronounced accumulation of apoptotic cells (ACs). Therefore, we hypothesized that SCARF1 will be important for clearance of ACs and maintenance of self-tolerance in humans, and that dysregulation of this process could contribute to SLE. In this article, we show that SCARF1 is highly expressed on phagocytic cells, where it functions as an efferocytosis receptor. In healthy individuals, we discovered that engagement of SCARF1 by ACs on BDCA1+ dendritic cells initiates an IL-10 anti-inflammatory response mediated by the phosphorylation of STAT1 and STAT3. Unexpectedly, there was no significant difference in SCARF1 expression in samples of patients with SLE compared with healthy donor samples. However, we detected anti-SCARF1 autoantibodies in 26% of patients with SLE, which was associated with dsDNA Ab positivity. Furthermore, our data show a direct correlation of the levels of anti-SCARF1 in the serum and defects in the removal of ACs. Depletion of Ig restores efferocytosis in SLE serum, suggesting that defects in the removal of ACs are partially mediated by SCARF1 pathogenic autoantibodies. Our data demonstrate that human SCARF1 is an AC receptor in dendritic cells and plays a role in maintaining tolerance and homeostasis.

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Dysregulated Pulmonary Inflammatory Responses Exacerbate the Outcome of Secondary Aspergillosis Following Influenza.

Inhalation of airborne conidia of the ubiquitous fungus Aspergillus fumigatus commonly occurs but invasive aspergillosis is rare except in profoundly immunocompromised persons. Severe influenza predisposes patients to invasive pulmonary aspergillosis by mechanisms that are poorly defined. Using a post-influenza aspergillosis model, we found that superinfected mice had 100% mortality when challenged with A. fumigatus conidia on days 2 and 5 (early stages) of influenza A virus infection but 100% survival when challenged on days 8 and 14 (late stages). Influenza-infected mice superinfected with A. fumigatus had increased levels of the pro-inflammatory cytokines and chemokines IL-6, TNFα, IFNß, IL-12p70, IL-1α, IL-1ß, CXCL1, G-CSF, MIP-1α, MIP-1ß, RANTES and MCP-1. Surprisingly, on histopathological analysis, superinfected mice did not have greater lung inflammation compared with mice infected with influenza alone. Mice infected with influenza had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus , but only if the fungal challenge was executed during the early stages of influenza infection. However, influenza infection did not have a major effect on neutrophil phagocytosis and killing of A. fumigatus conidia. Moreover, minimal germination of conidia was seen on histopathology even in the superinfected mice. Taken together, our data suggest that the high mortality rate seen in mice during the early stages of influenza-associated pulmonary aspergillosis is multifactorial, with a greater contribution from dysregulated inflammation than microbial growth. Importance: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by A. fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus , influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Dysregulated pulmonary inflammatory responses exacerbate the outcome of secondary aspergillosis following influenza.

IMPORTANCE: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by Aspergillus fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model of IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Current Insights in Cutaneous Lupus Erythematosus Immunopathogenesis.

Cutaneous Lupus Erythematosus (CLE) is a clinically diverse group of autoimmune skin diseases with shared histological features of interface dermatitis and autoantibodies deposited at the dermal-epidermal junction. Various genetic and environmental triggers of CLE promote infiltration of T cells, B cells, neutrophils, antigen presenting cells, and NK cells into lesional skin. In this mini-review, we will discuss the clinical features of CLE, insights into CLE immunopathogenesis, and novel treatment approaches.

Toll-like receptor 9-dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA.

Phagocytic defenses are critical for effective host defenses against the opportunistic fungal pathogen Aspergillus fumigatus. Previous studies found that following challenge with A. fumigatus, Toll-like receptor 9 (TLR9) knockout mice survived longer than wild-type mice. However, the mechanism responsible was not defined. Here we demonstrate that A. fumigatus contains unmethylated CpG sequences, the natural ligands for TLR9. A. fumigatus DNA and synthetic CpG-rich oligodeoxynucleotides (ODNs) containing sequences found in the A. fumigatus genome potently stimulated the production of proinflammatory cytokines in mouse bone marrow-derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells. The response was decreased when the fungal DNA was treated with a CpG methylase or with CpG-specific endonucleases. A role for TLR9 was demonstrated as cytokine production was abolished in BMDCs from TLR9-deficient mice. Moreover, transfection of HEK293 cells with human TLR9 conferred responsiveness to synthetic CpG-rich ODNs containing sequences found in A. fumigatus DNA. Taken together, these data demonstrate that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines, which may contribute to the immune response to the pathogen.

The role of dendritic cells in the innate recognition of pathogenic fungi (A. fumigatus, C. neoformans and C. albicans).

Dendritic cells (DCs) are the bridge between the innate and adaptive immune system. DCs are responsible for sensing and patrolling the environment, initiating a host response and instructing the proper adaptive immune response against pathogens. Recent advances in medical treatments have led to increased use of immunosuppressive drugs, leading to the emergence of fungal species that cause life-threatening infections in humans. Three of these opportunistic fungal pathogens: Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans pose the biggest concern for the immune-compromised host. Here we will review the interactions between DCs and these fungal pathogens, the receptors expressed on DCs that mediate these responses and the signaling mechanisms that shape the adaptive host response.

A nonredundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen Aspergillus fumigatus.

While plasmacytoid dendritic cells (pDCs), a natural type I interferon (IFN)-producing cell type, are regarded as critical for innate immunity to viruses, their role in defense against fungal infections remains unknown. We examined the interactions of pDCs with hyphae of the invasive human fungal pathogen Aspergillus fumigatus. Human pDCs spread over hyphae and inhibited their growth. Antifungal activity was retained in pDC lysates, did not require direct fungal contact, and was partially reversed by zinc. Incubation with hyphae resulted in pDC cytotoxicity, partly due to fungal gliotoxin secretion. Following hyphal stimulation, pDCs released proinflammatory cytokines via a TLR9-independent mechanism. Pulmonary challenge of mice with A. fumigatus resulted in a substantial influx of pDCs into lungs, and pDC-depleted mice were hypersusceptible to invasive aspergillosis. These data demonstrate the antifungal activity of pDCs against A. fumigatus and establish their nonredundant role in host defenses against invasive aspergillosis in vivo.

Discovering innate immunity genes using differential display: a story of RNA helicases.

In this review we discuss the current literature for RNA helicases in response to RNA virus infection. We show the use of Differential Display Reverse Transcription PCR methodology (DD) to analyze virus-host interactions and we present current findings in dengue virus-induced gene expression of RNA helicases.