Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Harnessing endogenous peptide compounds as potential therapeutics for severe influenza.

BACKGROUND: Excessive pulmonary inflammation and damage are characteristic features of severe influenza virus infections. LAT8881 is a synthetic, 16 amino acid cyclic peptide form of a naturally occurring C-terminal fragment of human growth hormone with therapeutic efficacy against influenza. Shorter, linear peptides are typically easier to manufacture and formulate for delivery than larger cyclic peptides. A 6 amino acid linear peptide fragment of LAT8881, LAT9997, was investigated as a potential influenza therapy. METHODS: LAT9997 was evaluated for its potential to limit disease in a preclinical mouse model of severe influenza infection. RESULTS: Intranasal treatment of mice with either LAT8881 or LAT9997 from day 1 following influenza infection significantly improved survival outcomes. Initiating LAT9997 treatment at the onset of severe disease, also significantly improved disease severity. Greater disease resistance in LAT9997-treated mice correlated with reduced lung immunopathology, damage markers, vascular leak, and epithelial cell death. Treatment reduced viral loads, cytokines, and neutrophil infiltration in the airways, yet maintained protective alveolar macrophages in a dose-dependent manner. Sequential trimming of N- and C-terminal amino acids from LAT9997 revealed a structure-activity relationship. CONCLUSIONS: These findings provide preclinical evidence that therapeutic LAT9997 treatment limits viral burden and characteristic features of severe influenza, including hyperinflammation and lung damage.

A high-dimensional cytometry atlas of peripheral blood over the human life span.

Age can profoundly affect susceptibility to a broad range of human diseases. Children are more susceptible to some infectious diseases such as diphtheria and pertussis, while in others, such as coronavirus disease 2019 and hepatitis A, they are more protected compared with adults. One explanation is that the composition of the immune system is a major contributing factor to disease susceptibility and severity. While most studies of the human immune system have focused on adults, how the immune system changes after birth remains poorly understood. Here, using high-dimensional spectral flow cytometry and computational methods for data integration, we analyzed more than 50 populations of immune cells in the peripheral blood, generating an immune cell atlas that defines the healthy human immune system from birth up to 75 years of age. We focused our efforts on children under 18 years old, revealing major changes in immune cell populations after birth and in children of schooling age. Specifically, CD4+ T effector memory cells, Vδ2+ gamma delta (γδ)T cells, memory B cells, plasmablasts, CD11c+ B cells and CD16+ CD56bright natural killer (NK) cells peaked in children aged 5-9 years old, whereas frequencies of T helper 1, T helper 17, dendritic cells and CD16+ CD57+ CD56dim NK cells were highest in older children (10-18 years old). The frequency of mucosal-associated invariant T cells was low in the first several years of life and highest in adults between 19 and 30 years old. Late adulthood was associated with fewer mucosal-associated invariant T cells and Vδ2+ γδ T cells but with increased frequencies of memory subsets of B cells, CD4+ and CD8+ T cells and CD57+ NK cells. This human immune cell atlas provides a critical resource to understand changes to the immune system during life and provides a reference for investigating the immune system in the context of human disease. This work may also help guide future therapies that target specific populations of immune cells to protect at-risk populations.

Interleukin-1ß exacerbates disease and is a potential therapeutic target to reduce pulmonary inflammation during severe influenza A virus infection.

Hyperinflammatory responses including the production of NLRP3-dependent interleukin (IL)-1ß is a characteristic feature of severe and fatal influenza A virus (IAV) infections. The NLRP3 inflammasome has been shown to play a temporal role during severe IAV immune responses, with early protective and later detrimental responses. However, the specific contribution of IL-1ß in modulating IAV disease in vivo is currently not well defined. Here, we identified that activation of NLRP3-dependent IL-1ß responses occurs rapidly following HKx31 H3N2 infection, prior to the onset of severe IAV disease. Mature IL-1ß was detectable in vivo in both hemopoietic and nonhemopoietic cells. Significantly, therapeutic inhibition of IL-1ß in the airways with intranasal anti-IL-1ß antibody treatment from day 3 postinfection, corresponding to the onset of clinical signs of disease, significantly prolonged survival and reduced inflammation in the airways. Importantly, early targeting of IL-1ß from day 1 postinfection also improved survival. Together, these studies specifically define a role for IL-1ß in contributing to the development of hyperinflammation and disease and indicate that targeting IL-1ß is a potential therapeutic strategy for severe IAV infections.

Classical Type 1 Dendritic Cells Dominate Priming of Th1 Responses to Herpes Simplex Virus Type 1 Skin Infection.

CD4+ T cell responses are crucial for the control of many intracellular pathogens, yet the requirements for their induction are not fully understood. To better understand the role that various dendritic cell (DC) subtypes play in CD4+ T cell priming, we compared in vivo T cell responses to skin inoculation of mice with infectious or UV-inactivated HSV type 1. Localized infection elicited a Th1 response that was primed in skin-draining lymph nodes involving Ag presentation by migratory dermal and lymph node-resident DC. However, expansion and Th1 differentiation was impaired in response to UV-inactivated virus (UV-HSV), and this defect correlated with a restriction of Ag presentation to migratory CD103- dermal DC. A similar differentiation defect was seen in infected mice lacking CD8α+ and CD103+ classical type 1 DC (cDC1). Finally, Th1 differentiation after UV-HSV inoculation was rescued by targeted Ag delivery to CD8α+ and CD103+ cDC1 using an anti-Clec9A Ab construct. This suggests that Ag presentation by cDC1 is crucial for optimal Th1 immunity to HSV type 1 infection and potentially other pathogens of the skin.

Limited Internodal Migration of T Follicular Helper Cells after Peripheral Infection with Herpes Simplex Virus-1.

The ability of CD4 T cells to give rise to specialized T follicular helper cells (TFH) critical to initiating appropriate Ab responses is regulated by environmental cues in lymphoid tissues draining the site of infection. In this study, we used a skin infection with HSV-1 characterized by the successive involvement of interconnected but distinct lymph nodes (LNs), to investigate the anatomical diversification of virus-specific CD4 T cell responses and the migratory capacity of TFH or their precursors. Whereas Th1 effector CD4 T cells expressing peripheral-targeting migration molecules readily migrated from primary to secondary reactive LNs, Bcl6(+) CXCR5(+) PD1(hi) TFH were largely retained at the site of initial activation with little spillover into the downstream LNs involved at later stages of infection. Consistent with this, TFH maintained high-level surface expression of CD69, indicative of impaired migratory capacity. Notably, the biased generation and retention of TFH in primary LNs correlated with a preferential generation of germinal centers at this site. Our results highlight a limited anatomical diversification of TFH responses and germinal center reactions that were imprinted within the first few cell divisions during TFH differentiation in LNs draining the site of initial infection.

Killer cell immunoglobulin-like receptor 3DL1-mediated recognition of human leukocyte antigen B.

Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0-D1-D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards ß2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an 'innate HLA sensor' domain. Second, whereas the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA-B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species.

Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells.

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.

A structural basis for antigen presentation by the MHC class Ib molecule, Qa-1b.

The primary function of the monomorphic MHC class Ib molecule Qa-1(b) is to present peptides derived from the leader sequences of other MHC class I molecules for recognition by the CD94-NKG2 receptors expressed by NK and T cells. Whereas the mode of peptide presentation by its ortholog HLA-E, and subsequent recognition by CD94-NKG2A, is known, the molecular basis of Qa-1(b) function is unclear. We have assessed the interaction between Qa-1(b) and CD94-NKG2A and shown that they interact with an affinity of 17 µM. Furthermore, we have determined the structure of Qa-1(b) bound to the leader sequence peptide, Qdm (AMAPRTLLL), to a resolution of 1.9 Å and compared it with that of HLA-E. The crystal structure provided a basis for understanding the restricted peptide repertoire of Qa-1(b). Whereas the Qa-1(b-AMAPRTLLL) complex was similar to that of HLA-E, significant sequence and structural differences were observed between the respective Ag-binding clefts. However, the conformation of the Qdm peptide bound by Qa-1(b) was very similar to that of peptide bound to HLA-E. Although a number of conserved innate receptors can recognize heterologous ligands from other species, the structural differences between Qa-1(b) and HLA-E manifested in CD94-NKG2A ligand recognition being species specific despite similarities in peptide sequence and conformation. Collectively, our data illustrate the structural homology between Qa-1(b) and HLA-E and provide a structural basis for understanding peptide repertoire selection and the specificity of the interaction of Qa-1(b) with CD94-NKG2 receptors.

Naturally derived cytokine peptides limit virus replication and severe disease during influenza A virus infection.

Objectives: Novel host-targeted therapeutics could treat severe influenza A virus (IAV) infections, with reduced risk of drug resistance. LAT8881 is a synthetic form of the naturally occurring C-terminal fragment of human growth hormone. Acting independently of the growth hormone receptor, it can reduce inflammation-induced damage and promote tissue repair in an animal model of osteoarthritis. LAT8881 has been assessed in clinical trials for the treatment of obesity and neuropathy and has an excellent safety profile. We investigated the potential for LAT8881, its metabolite LAT9991F and LAT7771 derived from prolactin, a growth hormone structural homologue, to treat severe IAV infection. Methods: LAT8881, LAT9991F and LAT7771 were evaluated for their effects on cell viability and IAV replication in vitro, as well as their potential to limit disease in a preclinical mouse model of severe IAV infection. Results: In vitro LAT8881 treatment enhanced cell viability, particularly in the presence of cytotoxic stress, which was countered by siRNA inhibition of host lanthionine synthetase C-like proteins. Daily intranasal treatment of mice with LAT8881 or LAT9991F, but not LAT7771, from day 1 postinfection significantly improved influenza disease resistance, which was associated with reduced infectious viral loads, reduced pro-inflammatory cytokines and increased abundance of protective alveolar macrophages. LAT8881 treatment in combination with the antiviral oseltamivir phosphate led to more pronounced reduction in markers of disease severity than treatment with either compound alone. Conclusion: These studies provide the first evidence identifying LAT8881 and LAT9991F as novel host-protective therapies that improve survival, limit viral replication, reduce local inflammation and curtail tissue damage during severe IAV infection. Evaluation of LAT8881 and LAT9991F in other infectious and inflammatory conditions of the airways is warranted.

Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection.

Excessive inflammation and tissue damage during severe influenza A virus (IAV) infection can lead to the development of fatal pulmonary disease. Pyroptosis is a lytic and pro-inflammatory form of cell death executed by the pore-forming protein gasdermin D (GSDMD). In this study, we investigated a potential role for GSDMD in promoting the development of severe IAV disease. IAV infection resulted in cleavage of GSDMD in vivo and in vitro in lung epithelial cells. Mice genetically deficient in GSDMD (Gsdmd-/-) developed less severe IAV disease than wildtype mice and displayed improved survival outcomes. GSDMD deficiency significantly reduced neutrophil infiltration into the airways as well as the levels of pro-inflammatory cytokines TNF, IL-6, MCP-1, and IL-1α and neutrophil-attracting chemokines CXCL1 and CXCL2. In contrast, IL-1ß and IL-18 responses were not largely impacted by GSDMD deficiency. In addition, Gsdmd-/- mice displayed significantly improved influenza disease resistance with reduced viral burden and less severe pulmonary pathology, including decreased epithelial damage and cell death. These findings indicate a major role for GSDMD in promoting damaging inflammation and the development of severe IAV disease.

Unconventional T Cell Immunity in the Lungs of Young Children with Cystic Fibrosis.

BACKGROUND: People with Cystic Fibrosis (CF) develop pulmonary inflammation, chronic infection and structural lung damage early in life, with these manifestations being prevalent among preschool children and infants. While early immune events are believed to play critical roles in shaping the progression, severity and disease burden later in life, T cells and their subsets are poorly studied in the CF lung, particularly during the formative early stages of disease. METHODS: Using flow cytometry, we analyzed Mucosal Associated Invariant T (MAIT) cells, γδ T cells, and Natural Killer T (NKT)-like cells in bronchoalveolar lavage (BAL) samples from seventeen children with CF, aged two to six years old. The effect of age, sex and lung infections on the frequencies of these cells in BAL samples was analysed (grouped data were tested for normality and compared by t-test or Kruskal-Wallis analysis). RESULTS: No difference was noted in the proportions of unconventional T cells related to the sex or age of the children. The frequency of γδ T cells and MAIT cells appeared unchanged by infection status. However, viral infections were associated with a significant increase in the proportion of NKT-like cells. CONCLUSIONS: By evaluating T cells in the lungs of children during the early formative stages of CF, this study identified potentially important interactions between these cells and viral pathogens.

Too young to die? How aging affects cellular innate immune responses to influenza virus and disease severity.

Influenza is a respiratory viral infection that causes significant morbidity and mortality worldwide. The innate immune cell response elicited during influenza A virus (IAV) infection forms the critical first line of defense, which typically is impaired as we age. As such, elderly individuals more commonly succumb to influenza-associated complications, which is reflected in most aged animal models of IAV infection. Here, we review the important roles of several major innate immune cell populations in influenza pathogenesis, some of which being deleterious to the host, and the current knowledge of how age-associated numerical, phenotypic and functional cell changes impact disease development. Further investigation into age-related modulation of innate immune cell responses, using appropriate animal models, will help reveal how immunity to IAV may be compromised by aging and inform the development of novel therapies, tailored for use in this vulnerable group.

Immune Profiling of Cord Blood From Preterm and Term Infants Reveals Distinct Differences in Pro-Inflammatory Responses.

Background: Preterm infants are highly vulnerable to infectious disease. While many factors are likely to contribute to this enhanced susceptibility, the immature nature of the preterm immune system is postulated as one key factor. Methods: In our study, we used high-dimensional flow cytometry and cytokine assays to characterise the immune profiles in 25 preterm (range: 30.4-34.1 weeks gestational age) and 25 term infant (range: 37-40 weeks gestational age) cord blood samples. Results: We found that preterm infants exhibit reduced frequencies of monocytes, CD56bright NK cells, CD8+ T-cells, γδ T-cells and an increased frequency of intermediate monocytes, CD4+ T-cells, central memory CD4+ and CD8+ T-cells, Tregs and transitional B-cells compared to term infants. Pro-inflammatory cytokines IL-1ß, IL-6 and IL-17A were lower in preterm infants in addition to chemokines IL-8, eotaxin, MIP-1α and MIP-1ß. However, IL-15 and MCP-1 were higher in preterm infants. Conclusion: Overall, we identify key differences in pro-inflammatory immune profiles between preterm and term infants. These findings may help to explain why preterm infants are more susceptible to infectious disease during early life and facilitate the development of targeted interventions to protect this highly vulnerable group.

Natural killer cell receptors regulate responses of HLA-E-restricted T cells.

Human cytomegalovirus (CMV) infection can stimulate robust human leukocyte antigen (HLA)-E-restricted CD8+ T cell responses. These T cells recognize a peptide from UL40, which differs by as little as a single methyl group from self-peptides that also bind HLA-E, challenging their capacity to avoid self-reactivity. Unexpectedly, we showed that the UL40/HLA-E T cell receptor (TCR) repertoire included TCRs that had high affinities for HLA-E/self-peptide. However, paradoxically, lower cytokine responses were observed from UL40/HLA-E T cells bearing TCRs with high affinity for HLA-E. RNA sequencing and flow cytometric analysis revealed that these T cells were marked by the expression of inhibitory natural killer cell receptors (NKRs) KIR2DL1 and KIR2DL2/L3. On the other hand, UL40/HLA-E T cells bearing lower-affinity TCRs expressed the activating receptor NKG2C. Activation of T cells bearing higher-affinity TCRs was regulated by the interaction between KIR2D receptors and HLA-C. These findings identify a role for NKR signaling in regulating self/non-self discrimination by HLA-E-restricted T cells, allowing for antiviral responses while avoiding contemporaneous self-reactivity.

Enrichment of Cytomegalovirus-induced NKG2C+ Natural Killer Cells in the Lung Allograft.

BACKGROUND: In lung transplant recipients, immunosuppressive medications result in impaired antiviral immunity and a propensity for cytomegalovirus (CMV) reactivation within the lung allograft. Natural killer (NK) cells play a key role in immunity to CMV, with an increase in the proportion of NK cells expressing activating CD94-NKG2C receptors in the blood being a strong correlate of CMV infection. Whether a similar increase in NKG2C NK cells occurs in lung transplant recipients following CMV reactivation in the allograft and if such cells contribute to viral control remains unclear. METHODS: In this pilot study, we longitudinally assessed the frequency and phenotype of NKG2C NK cells in the blood and bronchoalveolar lavage (BAL) of lung transplant recipients and stratified recipients based on their risk of developing CMV disease. RESULTS: We observed an increase in the proportion of NKG2C NK cells in the blood and BAL of CMV high-risk patients, coincident with both the cessation of antiviral prophylaxis and subsequent detection of actively replicating CMV in the blood and lung allograft. Additionally, these NKG2C NK cells expressed killer-cell immunoglobulin-like receptors distinct from those of other NK subsets and BAL NKG2C NK cells possessed an activated phenotype. Finally, the frequency of NKG2C NK cells in the BAL may be inversely correlated with CMV blood titers. CONCLUSIONS: Monitoring the phenotype of NK cells postlung transplant may be a useful biomarker for monitoring patient levels of CMV immunity.