Leukocyte surface expression of the endoplasmic reticulum chaperone GRP78 is increased in severe COVID-19.

Hyperinflammation present in individuals with severe COVID-19 has been associated with an exacerbated cytokine production and hyperactivated immune cells. Endoplasmic reticulum stress leading to the unfolded protein response has been recently reported as an active player in inducing inflammatory responses. Once unfolded protein response is activated, GRP78, an endoplasmic reticulum-resident chaperone, is translocated to the cell surface (sGRP78), where it is considered a cell stress marker; however, its presence has not been evaluated in immune cells during disease. Here we assessed the presence of sGRP78 on different cell subsets in blood samples from severe or convalescent COVID-19 patients. The frequency of CD45+sGRP78+ cells was higher in patients with the disease compared to convalescent patients. The latter showed similar frequencies to healthy controls. In patients with COVID-19, the lymphoid compartment showed the highest presence of sGRP78+ cells versus the myeloid compartment. CCL2, TNF-α, C-reactive protein, and international normalized ratio measurements showed a positive correlation with the frequency of CD45+sGRP78+ cells. Finally, gene expression microarray data showed that activated T and B cells increased the expression of GRP78, and peripheral blood mononuclear cells from healthy donors acquired sGRP78 upon activation with ionomycin and PMA. Thus, our data highlight the association of sGRP78 on immune cells in patients with severe COVID-19.

Clinical and Immunological Factors That Distinguish COVID-19 From Pandemic Influenza A(H1N1).

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a global health threat with the potential to cause severe disease manifestations in the lungs. Although COVID-19 has been extensively characterized clinically, the factors distinguishing SARS-CoV-2 from other respiratory viruses are unknown. Here, we compared the clinical, histopathological, and immunological characteristics of patients with COVID-19 and pandemic influenza A(H1N1). We observed a higher frequency of respiratory symptoms, increased tissue injury markers, and a histological pattern of alveolar pneumonia in pandemic influenza A(H1N1) patients. Conversely, dry cough, gastrointestinal symptoms and interstitial lung pathology were observed in COVID-19 cases. Pandemic influenza A(H1N1) was characterized by higher levels of IL-1RA, TNF-α, CCL3, G-CSF, APRIL, sTNF-R1, sTNF-R2, sCD30, and sCD163. Meanwhile, COVID-19 displayed an immune profile distinguished by increased Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokine levels, along with IL-1ß, IL-6, CCL11, VEGF, TWEAK, TSLP, MMP-1, and MMP-3. Our data suggest that SARS-CoV-2 induces a dysbalanced polyfunctional inflammatory response that is different from the immune response against pandemic influenza A(H1N1). Furthermore, we demonstrated the diagnostic potential of some clinical and immune factors to differentiate both diseases. These findings might be relevant for the ongoing and future influenza seasons in the Northern Hemisphere, which are historically unique due to their convergence with the COVID-19 pandemic.

Corrigendum: CXCL17 Is a Specific Diagnostic Biomarker for Severe Pandemic Influenza A(H1N1) That Predicts Poor Clinical Outcome.

[This corrects the article DOI: 10.3389/fimmu.2021.633297.].

CXCL17 Is a Specific Diagnostic Biomarker for Severe Pandemic Influenza A(H1N1) That Predicts Poor Clinical Outcome.

The C-X-C motif chemokine ligand 17 (CXCL17) is chemotactic for myeloid cells, exhibits bactericidal activity, and exerts anti-viral functions. This chemokine is constitutively expressed in the respiratory tract, suggesting a role in lung defenses. However, little is known about the participation of CXCL17 against relevant respiratory pathogens in humans. Here, we evaluated the serum levels and lung tissue expression pattern of CXCL17 in a cohort of patients with severe pandemic influenza A(H1N1) from Mexico City. Peripheral blood samples obtained on admission and seven days after hospitalization were processed for determinations of serum CXCL17 levels by enzyme-linked immunosorbent assay (ELISA). The expression of CXCL17 was assessed by immunohistochemistry (IHQ) in lung autopsy specimens from patients that succumbed to the disease. Serum CXCL17 levels were also analyzed in two additional comparative cohorts of coronavirus disease 2019 (COVID-19) and pulmonary tuberculosis (TB) patients. Additionally, the expression of CXCL17 was tested in lung autopsy specimens from COVID-19 patients. A total of 122 patients were enrolled in the study, from which 68 had pandemic influenza A(H1N1), 24 had COVID-19, and 30 with PTB. CXCL17 was detected in post-mortem lung specimens from patients that died of pandemic influenza A(H1N1) and COVID-19. Interestingly, serum levels of CXCL17 were increased only in patients with pandemic influenza A(H1N1), but not COVID-19 and PTB. CXCL17 not only differentiated pandemic influenza A(H1N1) from other respiratory infections but showed prognostic value for influenza-associated mortality and renal failure in machine-learning algorithms and regression analyses. Using cell culture assays, we also identified that human alveolar A549 cells and peripheral blood monocyte-derived macrophages increase their CXCL17 production capacity after influenza A(H1N1) pdm09 virus infection. Our results for the first time demonstrate an induction of CXCL17 specifically during pandemic influenza A(H1N1), but not COVID-19 and PTB in humans. These findings could be of great utility to differentiate influenza and COVID-19 and to predict poor prognosis specially at settings of high incidence of pandemic A(H1N1). Future studies on the role of CXCL17 not only in severe pandemic influenza, but also in seasonal influenza, COVID-19, and PTB are required to validate our results.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Tuning Subunit Vaccines with Novel TLR Triagonist Adjuvants to Generate Protective Immune Responses against Coxiella burnetii.

Coxiella burnetii is an obligate intracellular bacterium and the causative agent of Q fever. C. burnetii is considered a potential bioterrorism agent because of its low infectious dose; resistance to heat, drying, and common disinfectants; and lack of prophylactic therapies. Q-Vax, a formalin-inactivated whole-bacteria vaccine, is currently the only prophylactic measure that is protective against C. burnetii infections but is not U.S. Food and Drug Administration approved. To overcome the safety concerns associated with the whole-bacteria vaccine, we sought to generate and evaluate recombinant protein subunit vaccines against C. burnetii To accomplish this, we formulated C. burnetii Ags with a novel TLR triagonist adjuvant platform, which used combinatorial chemistry to link three different TLR agonists together to form one adjuvanting complex. We evaluated the immunomodulatory activity of a panel of TLR triagonist adjuvants and found that they elicited unique Ag-specific immune responses both in vitro and in vivo. We evaluated our top candidates in a live C. burnetii aerosol challenge model in C56BL/6 mice and found that several of our novel vaccine formulations conferred varying levels of protection to the challenged animals compared with sham immunized mice, although none of our candidates were as protective as the commercial vaccine across all protection criteria that were analyzed. Our findings characterize a novel adjuvant platform and offer an alternative approach to generating protective and effective vaccines against C. burnetii.

Linked Toll-Like Receptor Triagonists Stimulate Distinct, Combination-Dependent Innate Immune Responses.

Traditional vaccination strategies have failed to generate effective vaccines for many infections like tuberculosis and HIV. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like receptors (TLRs). Vaccines with multiple TLRs as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization-a critical feature of whole-cell vaccines. Second, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo. The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-κB, ISGs), cytokine profiles (IL12-p70, TNF-α, IL-6, IL-10, CCL2, IFN-α, IFN-ß, IFN-γ), and in vivo cytokine serum levels (IL-6, TNF-α, IL12-p40, IFN-α, IFN-ß). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more Th1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward TH2 biasing profiles despite the presence of TLR9. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development.

CRTAM Shapes the Gut Microbiota and Enhances the Severity of Infection.

Gut lymphocytes and the microbiota establish a reciprocal relationship that impacts the host immune response. Class I-restricted T cell-associated molecule (CRTAM) is a cell adhesion molecule expressed by intraepithelial T cells and is required for their retention in the gut. In this study, we show that CRTAM expression affects gut microbiota composition under homeostatic conditions. Moreover, Crtam-/- mice infected with the intestinal pathogen Salmonella exhibit reduced Th17 responses, lower levels of inflammation, and reduced Salmonella burden, which is accompanied by expansion of other microbial taxa. Thus, CRTAM enhances susceptibility to Salmonella, likely by promoting the inflammatory response that promotes the pathogen's growth. We also found that the gut microbiota from wild-type mice, but not from Crtam-/- mice, induces CRTAM expression and Th17 responses in ex-germ-free mice during Salmonella infection. Our study demonstrates a reciprocal relationship between CRTAM expression and the gut microbiota, which ultimately impacts the host response to enteric pathogens.

Cxcl17-/- mice develop exacerbated disease in a T cell-dependent autoimmune model.

CXCL17 is a homeostatic chemokine in the mucosa known to chemoattract dendritic cells and macrophages but can also be expressed elsewhere under inflammatory conditions. Cxcl17-/- mice have lower numbers of macrophages or dendritic cells in mucosal tissues. CXCL17 is also able to chemoattract suppressor myeloid cells that can recruit regulatory T cells. To explore a possible role of Cxcl17 in T cells, we studied T cell populations from Cxcl17-/- or wild-type (WT) littermate mice. Cxcl17-/- mice have higher numbers of CD4+ and CD8+ T cells in spleen and lymph nodes (LNs). Upon activation, they produce higher levels of several proinflammatory cytokines and chemokines. Furthermore, a Cxcl17-/- mouse developed exacerbated disease in a T cell-dependent model of experimental autoimmune encephalomyelitis (EAE). By 18 days after immunization with myelin oligodendrocyte peptide, only 44% of Cxcl17-/- mice were still alive vs. 90% for WT mice. During EAE, Cxcl17-/- mice exhibited higher numbers of lymphoid and myeloid cells in spleen and LNs, whereas they had less myeloid cell infiltration in the CNS. Cxcl17-/- mice also had higher levels of some inflammatory cytokines in serum, suggesting that they may be involved in the poor survival of these mice. Abnormal T cell function may reflect altered myeloid cell migration, or it could be due to altered T cell development in the thymus. We conclude that CXCL17 is a novel factor regulating T cell homeostasis and function.

CCL28 Is Involved in Mucosal IgA Responses, Olfaction, and Resistance to Enteric Infections.

CCL28 is a mucosal chemokine that has been involved in various responses, including IgA production. We have analyzed its production in human tissues using a comprehensive microarray database. Its highest expression is in the salivary gland, indicating that it is an important component of saliva. It is also expressed in the trachea, bronchus, and in the mammary gland upon onset of lactation. We have also characterized a Ccl28-/- mouse that exhibits very low IgA levels in milk, and the IgA levels in feces are also reduced. These observations confirm a role for the CCL28/CCR10 chemokine axis in the recruitment of IgA plasmablasts to the lactating mammary gland. CCL28 is also expressed in the vomeronasal organ. We also detected olfactory defects (anosmia) in a Ccl28-/- mouse suggesting that CCL28 is involved in the function/development of olfaction. Importantly, Ccl28-/- mice are highly susceptible to Salmonella enterica serovar Typhimurium in an acute model of infection, indicating that CCL28 plays a major role in innate immunity against Salmonella in the gut. Finally, microbiome studies revealed modest differences in the gut microbiota between Ccl28-/- mice and their cohoused wild-type littermates. The latter observation suggests that under homeostatic conditions, CCL28 plays a limited role in shaping the gut microbiome.

Meteorin-like/Meteorin-ß Is a Novel Immunoregulatory Cytokine Associated with Inflammation.

We have described a novel cytokine encoded by a gene called Meteorin-like (Metrnl). Metrnl is a small (∼28 kDa) secreted protein expressed by activated macrophages and barrier tissues (mucosa and skin). Metrnl production by bone marrow macrophages is induced by several cytokines including TNF-α, IL-17α, IL-12, and IL-4 and inhibited by IFN-γ and TGF-ß. Metrnl expression in macrophages is also induced by LPS, and its levels in circulation are associated with inflammatory responses in vivo. Furthermore, Metrnl regulates the production of several cytokines and chemokines in macrophages. We have produced a Metrnl-/- mouse, which is viable and shows normal development. However, it exhibits dysregulated cytokine production, alterations in IgG production, and is highly susceptible to LPS in a sepsis model. Furthermore, older Metrnl-/- mice develop inflammatory lesions, suggesting that Metrnl participates in the control of inflammatory responses. Taken together, these observations indicate that Metrnl encodes a novel immunoregulatory cytokine associated with inflammatory responses that we have designated Meteorin-ß.

Interleukin 30 to Interleukin 40.

Cytokines are important molecules that regulate the ontogeny and function of the immune system. They are small secreted proteins usually produced upon activation of cells of the immune system, including lymphocytes and myeloid cells. Many cytokines have been described, and several have been recognized as pivotal players in immune responses and in human disease. In fact, several anticytokine antibodies have proven effective therapeutics, especially in various autoimmune diseases. In the last 15 years, new cytokines have been described, and many remain poorly understood. Among the most recent cytokines discovered are interleukins-30 (IL-30) to IL-40. Several of these are members of other cytokine superfamilies, including several IL-1 superfamily members (IL-33, IL-36, IL-37, and IL-38) as well as several new members of the IL-12 family (IL-30, IL-35, and IL-39). The rest (IL-31, IL-32, IL-34, and IL-40) are encoded by genes that do not belong to any cytokine superfamily. Our aim of this review was to present a concise version of the information available on these novel cytokines to facilitate their understanding by members of the immunological community.

CXCL17 Chemokine-Dependent Mobilization of CXCR8+CD8+ Effector Memory and Tissue-Resident Memory T Cells in the Vaginal Mucosa Is Associated with Protection against Genital Herpes.

Circulating conventional memory CD8+ T cells (i.e., the CD8+ effector memory T [TEM] cell and CD8+ central memory T [TCM] cell subsets) and the noncirculating CD8+ tissue-resident memory T (TRM) cell subset play a critical role in mucosal immunity. Mucosal chemokines, including the recently discovered CXCL17, are also important in mucosal immunity because they are homeostatically expressed in mucosal tissues. However, whether the CXCL17 chemokine contributes to the mobilization of memory CD8+ T cell subsets to access infected mucosal tissues remains to be elucidated. In this study, we report that after intravaginal HSV type 1 infection of B6 mice, we detected high expression levels of CXCL17 and increased numbers of CD44highCD62LlowCD8+ TEM and CD103highCD8+ TRM cells expressing CXCR8, the cognate receptor of CXCL17, in the vaginal mucosa (VM) of mice with reduced genital herpes infection and disease. In contrast to wild-type B6 mice, the CXCL17-/- mice developed 1) fewer CXCR8+CD8+ TEM and TRM cells associated with more virus replication in the VM and more latency established in dorsal root ganglia, and 2) reduced numbers and frequencies of functional CD8+ T cells in the VM. These findings suggest that the CXCL17/CXCR8 chemokine pathway plays a crucial role in mucosal vaginal immunity by promoting the mobilization of functional protective CD8+ TEM and CD8+ TRM cells, within this site of acute and recurrent herpes infection.

Chemokine ligand-receptor interactions critically regulate cutaneous wound healing.

BACKGROUND: Wound healing represents a dynamic process involving directional migration of different cell types. Chemokines, a family of chemoattractive proteins, have been suggested to be key players in cell-to-cell communication and essential for directed migration of structural cells. Today, the role of the chemokine network in cutaneous wound healing is not fully understood. Unraveling the chemokine-driven communication pathways in this complex process could possibly lead to new therapeutic strategies in wound healing disorders. METHODS: We performed a systematic, comprehensive time-course analysis of the expression and function of a broad variety of cytokines, growth factors, adhesion molecules, matrixmetalloproteinases and chemokines in a murine cutaneous wound healing model. RESULTS: Strikingly, chemokines were found to be among the most highly regulated genes and their expression was found to coincide with the expression of their matching receptors. Accordingly, we could show that resting and activated human primary keratinocytes (CCR3, CCR4, CCR6, CXCR1, CXCR3), dermal fibroblasts (CCR3, CCR4, CCR10) and dermal microvascular endothelial cells (CCR3, CCR4, CCR6, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3) express a distinct and functionally active repertoire of chemokine receptors. Furthermore, chemokine ligand-receptor interactions markedly improved the wound repair of structural skin cells in vitro. CONCLUSION: Taken together, we here present the most comprehensive analysis of mediators critically involved in acute cutaneous wound healing. Our findings suggest therapeutic approaches for the management of wound closure by targeting the chemokine network.

Identification of IL-40, a Novel B Cell-Associated Cytokine.

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99), that is expressed in bone marrow and fetal liver and whose expression is also induced in peripheral B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (∼27-kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer Peyer's patches and lower numbers of IgA-secreting cells. The microbiome of C17orf99-/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. Although naive B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL-4 and TGF-ß1, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell-associated cytokine, which we have called IL-40, that plays an important role in humoral immune responses and may also play a role in B cell development. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observations suggest that it may play a role in the pathogenesis of certain human diseases.

Omics techniques and biobanks to find new biomarkers for the early detection of acute lymphoblastic leukemia in middle-income countries: a perspective from Mexico / Técnicas ómicas y biobancos para el hallazgo de nuevos biomarcadores para la detección temprana de leucemia linfoblástica aguda en países de ingresos medios: una perspectiva mexicana

Abstract: Acute lymphoblastic leukemia (ALL) affects the quality of life of many children in the world and particularly in Mexico, where a high incidence has been reported. With a proper financial investment and with well-organized institutions caring for those patients, together with solid platforms to perform high-throughput analyses, we propose the creation of a Mexican repository system of serum and cells from bone marrow and blood samples derived from tissues of pediatric patients with ALL diagnosis. This resource, in combination with omics technologies, particularly proteomics and metabolomics, would allow longitudinal studies, offering an opportunity to design and apply personalized ALL treatments. Importantly, it would accelerate the development of translational science and will lead us to further discoveries, including the identification of new biomarkers for the early detection of leukemia.

Resumen: La leucemia linfoblástica aguda (LLA) afecta la calidad de vida de una gran cantidad de individuos en edad pediátrica en todo el mundo; particularmente en México, donde se ha reportado una alta incidencia. Con un apropiado fondo de inversión financiera, así como instituciones adecuadamente organizadas al cuidado de los pacientes con LLA, en conjunto con plataformas sólidas para llevar a cabo análisis globales y de alto rendimiento, se propone la creación de un repositorio para la conservación de suero y células provenientes de médula ósea y sangre derivadas de pacientes pediátricos con LLA al diagnóstico. Estos recursos, en combinación con las tecnologías ómicas, en particular la proteómica y la metabolómica, podrían permitir el establecimiento de estudios longitudinales y ofrecer una oportunidad para el diseño y aplicación de tratamientos personalizados para la LLA. Esta estrategia permitiría acelerar el desarrollo de la ciencia traslacional, favoreciendo el hallazgo de importantes descubrimientos, incluyendo la identificación de nuevos biomarcadores para la detección temprana de la leucemia.

Transient Cannabinoid Receptor 2 Blockade during Immunization Heightens Intensity and Breadth of Antigen-specific Antibody Responses in Young and Aged mice.

The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.