Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs) and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of leukocytes via chemokine production.

Mast cells are among the first cells of our immune system to encounter exogenous danger. Intracellular receptors such as nucleotide-binding oligomerization domain (Nod) play an important role in responding to invading pathogens. Here, we have investigated the response of human mast cells to the Nod1 ligand M-TriDAP. Human cord blood-derived mast cells (CBMCs) were activated with M-TriDAP alone, or in combination with the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and zymosan. Release of pro-inflammatory chemokines and cytokines was measured by ELISA, cytometric bead array and LUMINEX, and degranulation was evaluated by analysis of histamine release. M-TriDAP induced a dose-dependent release of IL-8, MIP-1α, MIP-1β and TNF. In contrast, degranulation could not be observed. When cells were treated with M-TriDAP in combination with the TLR4 agonist LPS, but not with TLR2 agonist zymosan, the secretion of cytokines was augmented. We here present results demonstrating that human CBMCs are stimulated by the Nod1 agonist M-TriDAP alone and in combination with LPS to produce pro-inflammatory cytokines and chemokines. Our results add to the concept that mast cells constitute an important part of our host defense, as they are equipped with several types of important pattern recognition receptors, including TLRs and Nod.

The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.

Background: Mast cells respond to bacterial infection by producing mediators that recruit and activate leukocytes, mediate vasodilation and induce bronchoconstriction. These mast cell-driven responses play a crucial role in protective immunity against bacterial infection, but may contribute to bacterial exacerbation of allergic diseases. Bacterial components including peptidoglycan (PGN) and lipopeptides are known to activate receptors such as Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLR). Since the consequences of mast cell activation by individual or combinations of bacterial components have not been fully characterized, we determined the effects of TLR2 and NLR activation, alone or in combination, on human mast cell mediator production. Methods: Cord blood-derived human mast cells were activated by bacterial PGN, the lipopeptide Pam₃CSK₄ and NLR agonists alone or in combination. Mast cell degranulation, LTC₄ production and the production of cytokines were assessed. Results:PGN and the lipopeptide Pam₃CSK₄ induced human mast cells to produce the pro-inflammatory mediators IL-1β, IL-6, CXCL8 and LTC₄ in addition to anti-inflammatory IL-10. NLR agonists alone did not induce these responses, but significantly and selectively increased Pam₃CSK₄-mediated mast cell IL-6 production. PGN- and Pam₃CSK₄-induced mast cell IL-6, but not IL-1β, production was dependent on adenylyl cyclase activity and could be partially inhibited by the cyclooxygenase inhibitor naproxen. Conclusions: Increased mast cell IL-6 production in response to combined TLR2 and NLR activation could play a role in the protection against bacterial infection, but potentially exacerbate inflammation-dependent conditions. In addition, mast cell IL-6 production is dependent on adenylyl cyclase activity.

Background Prolidase deficiency (PD) is an autosomal recessive inborn multisystemic disease caused by mutations in the PEPD gene encoding the enzyme prolidase D, leading to defects in turnover of proline-containing proteins, such as collagen. PD is categorized as a metabolic disease, but also as an inborn error of immunity. PD presents with a range of findings including dysmorphic features, intellectual disabilities, recurrent infections, intractable skin ulceration, autoimmunity, and splenomegaly. Despite symptoms of immune dysregulation, only very limited immunologic assessments have been reported and standard therapies for PD have not been described. We report twin females with PD, including comprehensive immunologic profiles and treatment modalities used. Case presentation Patient 1 had recurrent infections in childhood. At age 13, she presented with telangiectasia, followed by painful, refractory skin ulcerations on her lower limbs, where skin biopsy excluded vasculitis. She had typical dysmorphic features of PD. Next-generation sequencing revealed pathogenic compound heterozygous mutations (premature stop codons) in the PEPD gene. Patient 2 had the same mutations, typical PD facial features, atopy, and telangiectasias, but no skin ulceration. Both patients had imidodipeptiduria. Lymphocyte subset analysis revealed low-normal frequency of Treg cells and decreased frequency of expression of the checkpoint molecule CTLA-4 in CD4+ TEM cells. Analysis of Th1, Th2, and Th17 profiles revealed increased inflammatory IL-17+ CD8+ TEM cells in both patients and overexpression of the activation marker HLA-DR on CD4+ TEM cells, reflecting a highly activated proinflammatory state. Neither PD patient had specific antibody deficiencies despite low CD4+CXCR5+ Tfh cells and low class-switched memory B cells. Plasma IL-18 levels were exceptionally high. Conclusions Immunologic abnormalities including skewed frequencies of activated inflammatory CD4+ and CD8+ TEM cells, decreased CTLA-4 expression, and defects in memory B cells may be a feature of immune dysregulation associated with PD; however, a larger sample size is required to validate these findings. The high IL-18 plasma levels suggest underlying autoinflammatory processes.

Mast cells are well known for their role in allergic inflammation where, upon aggregation of the high-affinity immunoglobulin E receptor, they release mediators such as histamine that cause classical allergic symptoms. Mast cells are located in almost all tissues and are especially numerous in organs that interface with the environment. Given this strategic location and the more recent notion that they are endowed with receptors that recognize endogenous and exogenous danger signals such as pathogens, it is not surprising that they function as important cells in immune surveillance. When mast cells are activated by pathogens they modulate innate and adaptive immune responses. In allergy, infections might cause exacerbation of the allergic reaction by affecting the reactivity of mast cells. With new developments within the field of mast cell biology, we will better understand how mast cells execute their effector functions. This knowledge will also help to improve the management of allergic diseases.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

ObjectiveRheumatoid arthritis (RA) is a chronic inflammatory disease mediated through complex immunologic pathways. Among RA patients receiving low‐dose methotrexate (MTX) monotherapy, approximately one‐half exhibit a meaningful clinical response within the first 6 months of starting treatment. Whether baseline immune phenotypes differ between subsequent MTX responders and nonresponders is unknown. This study utilized comprehensive T cell immunophenotyping to identify specific immunologic pathways associated with MTX‐nonresponsive joint inflammation in patients with RA.MethodsIn total, 32 patients with recent‐onset RA were treated with MTX therapy. After 6 months, 15 patients were categorized as responders and 17 as nonresponders. Comprehensive blood T cell immunophenotyping, using multiparameter immunofluorescence flow cytometry analyses, was performed at baseline and following 6 months of treatment.ResultsBaseline measures of disease activity (Disease Activity Score in 28 joints [DAS28], C‐reactive protein level, and erythrocyte sedimentation rate) did not differ between MTX responders and nonresponders following MTX treatment. Frequencies of CD4+ and CD8+ T cells were skewed to favor higher CD4:CD8 T cell ratios in MTX responders compared to nonresponders (P < 0.05). The proportion of inducible costimulator–expressing Treg cells was significantly greater among MTX nonresponders. Interleukin‐13 (IL‐13)–producing, but not interferon‐γ– or IL‐17–producing, CD4+ effector memory T (Tem) cells were significantly more frequent in MTX nonresponders (P < 0.05). The ratio of IL‐13+:IL‐17+ Tem cells among CD4+ Tem cells was 1.9‐fold higher in MTX nonresponders compared to responders (P < 0.05). Both the CD4:CD8 T cell ratio and the frequency of IL‐13+CD4+ Tem cells correlated with changes in the DAS28 score following MTX treatment, whereas T cell expression of immune checkpoint inhibitor markers (CTLA‐4, programmed death 1, and T cell immunoglobulin and mucin domain–containing protein 3) did not differ between MTX responders and nonresponders.ConclusionWe observed a bias toward type 2–polarized T cell inflammatory responses in the peripheral blood of MTX‐nonresponsive RA patients. Targeting the IL‐13+CD4+ T cell pathway could be a new therapeutic strategy in RA patients whose disease remains resistant to MTX.