Major Neutrophil-Derived Soluble Mediators Associate With Baseline Lung Pathology and Post-Treatment Recovery in Tuberculosis Patients

BackgroundThe inflammatory response to Mycobacterium tuberculosis results in variable degrees of lung pathology during active TB (ATB) with central involvement of neutrophils. Little is known about neutrophil-derived mediators and their role in disease severity at baseline and recovery upon TB treatment initiation.Methods107 adults with confirmed pulmonary TB were categorised based on lung pathology at baseline and following successful therapy using chest X-ray scores (Ralph scores) and GeneXpert bacterial load (Ct values). Plasma, sputum, and antigen-stimulated levels of MMP1, MMP3, MMP8, MMP9, MPO, S100A8/9, IL8, IL10, IL12/23(p40), GM-CSF, IFNγ, and TNF were analysed using multiplex cytokine arrays.ResultsAt baseline, neutrophil counts correlated with plasma levels of MMP8 (rho = 0.45, p = 2.80E−06), S100A8 (rho = 0.52, p = 3.00E−08) and GM-CSF (rho = 0.43, p = 7.90E−06). Levels of MMP8 (p = 3.00E−03), MMP1 (p = 1.40E−02), S100A8 (p = 1.80E−02) and IL12/23(p40) (p = 1.00E−02) were associated with severe lung damage, while sputum MPO levels were directly linked to lung damage (p = 1.80E−03), Mtb load (p = 2.10E−02) and lung recovery (p = 2.40E−02). Six months of TB therapy significantly decreased levels of major neutrophil-derived pro-inflammatory mediators: MMP1 (p = 4.90E−12 and p = 2.20E−07), MMP8 (p = 3.40E−14 and p = 1.30E−05) and MMP9 (p = 1.60E−04 and p = 1.50E−03) in plasma and sputum, respectively. Interestingly, following H37Rv whole cell lysate stimulation, S100A8 (p = 2.80E−02), MMP9 (p = 3.60E−02) and MPO (p = 9.10E−03) levels at month 6 were significantly higher compared to baseline. Sputum MMP1 (p = 1.50E−03), MMP3 (p = 7.58E−04), MMP9 (p = 2.60E−02) and TNF (p = 3.80E−02) levels were lower at month 6 compared to baseline in patients with good lung recovery.ConclusionIn this study, patients with severe lung pathology at baseline and persistent lung damage after treatment were associated with higher plasma and sputum levels of major pro-inflammatory neutrophil-derived mediators. Interestingly, low sputum MPO levels were associated with severe lung damage, higher Mtb burden and low recovery. Our data suggest that therapeutic agents which target these mediators should be considered for future studies on biomarkers and host-directed therapeutic approaches against TB-related lung pathology and/or lung recovery.

Immunomodulatory Properties of Blackberry Anthocyanins in THP-1 Derived Macrophages

An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.

An exploratory study demonstrating that salivary cytokine profiles are altered in children with small area thermal injury

Serum can be used to investigate changes in cytokine concentration following burn injury in children, however for children receiving treatment in an outpatient setting, blood is not routinely collected and therefore cannot be used for monitoring. The aim of this study was to investigate the use of saliva as a non-invasive tool for predicting burn outcomes by measuring the concentration of salivary cytokines in children with small area burns. A multiplex cytokine assay was used to measure 17 cytokines in the saliva of paediatric patients with burns (n = 20) and healthy controls (n = 20). After the removal of cytokines that had >30% of samples below the assay lower detection limit, six cytokines including IL-1β, IL-4, IL-7, IL-8, MCP-1 and TNFα were analysed for association with burns. IL-1β and IL-4 were found to be significantly elevated in the paediatric burn patients compared to healthy controls. Interestingly, IL-1β was also significantly elevated in scald burns, compared to contact burns. In addition, biologically meaningful differences in cytokine concentration were identified in patients with different burn characteristics, which warrant further investigation. This exploratory study provides evidence that cytokines can be detected in the saliva of children and that salivary cytokine profiles differ between healthy controls and children with burns. Overall, this study demonstrates the value of saliva for the investigation of cytokines and its potential application in paediatric diagnostics, specifically in situations where blood collection is not appropriate.

Gene signature and immune cell profiling by high-dimensional, single-cell analysis in COVID-19 patients, presenting Low T3 syndrome and coexistent hematological malignancies

Background Low T3 syndrome is frequent in patients admitted to intensive care units for critical illness and pneumonia. It has been reported also in patients with COVID-19, Hodgkin disease and chronic lymphocytic leukemia. We analyzed the clinical relevance of Low T3 syndrome in COVID-19 patients and, in particular, in those with associated hematological malignancies. Methods Sixty-two consecutive patients, hospitalized during the first wave of SARS-CoV-2 outbreak in Sant’Andrea University Hospital in Rome, were subdivided in 38 patients (Group A), showing low levels of FT3, and in 24 patients (Group B), with normal FT3 serum values. During the acute phase of the disease, we measured serum, radiologic and clinical disease severity markers and scores, in search of possible correlations with FT3 serum values. In addition, in 6 COVID-19 patients, 4 with Low T3 syndrome, including 2 with a hematological malignancy, and 2 with normal FT3 values, we performed, high-dimensional single-cell analysis by mass cytometry, multiplex cytokine assay and gene expression profiling in peripheral blood mononuclear cells (PBMC). Results Low FT3 serum values were correlated with increased Absolute Neutrophil Count, NLR and dNLR ratios and with reduced total count of CD3+, CD4+ and CD8+ T cells. Low FT3 values correlated also with increased levels of inflammation, tissue damage and coagulation serum markers as well as with SOFA, LIPI and TSS scores. The CyTOF analysis demonstrated reduction of the effector memory and terminal effector subtypes of the CD4+ T lymphocytes. Multiplex cytokine assay indicates that mainly IL-6, IP-10 and MCAF changes are associated with FT3 serum levels, particularly in patients with coexistent hematological malignancies. Gene expression analysis using Nanostring identified four genes differently expressed involved in host immune response, namely CD38, CD79B, IFIT3 and NLRP3. Conclusions Our study demonstrates that low FT3 serum levels are associated with severe COVID-19. Our multi-omics approach suggests that T3 is involved in the immune response in COVID-19 and coexistent hematological malignancy and new possible T3 target genes in these patients have been identified.

Profiling of cytokines, chemokines and growth factors in saliva and gingival crevicular fluid

In saliva and gingival crevicular fluid (GCF) soluble factors such as cytokines, chemokines and growth factors have shown a great potential serving as biomarkers for early detection and/or diagnosis of oral and systemic diseases. However, GCF and saliva, which one is a better source is still under debate. This study aimed to gain an overview of cytokines, chemokines and growth factors in saliva and GCF to pave the way for selecting suitable oral fluids for oral and systemic diseases. Multiplex cytokine assay was conducted to determine concentrations of cytokines, chemokines and growth factors in saliva and GCF samples from healthy subjects. The protocol for sample collection was carefully optimized. Stabilization, repeatability, and donor variation of the profiles were analyzed. We found that for different donors, cytokine and chemokine profiles showed unique patterns in saliva but similar patterns in GCF. In terms of growth factors, the profiles were individualized in saliva and GCF. All profiles stayed stable for the same healthy individual. In saliva, profiles of cytokines, chemokines and growth factors are individualized for different donors. In GCF, profiles of cytokines and chemokines are similar. Other factors, such as growth factors and T helper-related cytokines, are highly variable in donors. Profiles of soluble factors are not correlated in saliva and GCF. The comprehensive cytokine profiles in saliva and GCF reported in this work would serve as a good base for choosing promising cytokines for developing biomarkers in oral fluids.

Hypoxia Facilitates the Proliferation of Colorectal Cancer Cells by Inducing Cancer-associated Fibroblast-derived IL6.

Background: Colorectal cancer (CRC) is most common malignancy worldwide, and its underlying molecular mechanisms remain largely unexplored. Accumulating evidences indicate Cancer-Associated Fibroblasts (CAFs), abundant stromal cell population in the tumor microenvironment, play a key role in tumor development. Methods: We have successfully isolated CAFs and paired normal fibroblasts (NFs) from colorectal cancer tissues (n=10). By using multiplex cytokine profiling assay, we have identified IL-6 as a major cytokine released by CAFs. Coculturing of CAFs with CRC cell lines HCT116 or SW480 increase IL-6 release, and the secretion by CAFs can be further enhanced under hypoxia. By using CCK-8 assay, we have found HCT116 or SW480 cells treated with culture medium from CAFs, IL-6 or hypoxia showed a significant cell growth compared to control cells (P<0.01). Results: Mechanistically, we have found hypoxia can enhanced effect of IL-6/STAT3 signaling on CRC cells, in part, throughHIF-1a targets PKM2. Conclusions: In conclusion, our data clearly proposes the interconnected mechanisms for a constitutive activation of STAT3 signalby CAFs-derived IL-6 under hypoxia in colorectal cancer. The pharmacological inhibition of STAT3, PKM2 or HIF-1α can significantly reduce oncogenic effect of IL-6, providing a potential therapeutic target for CRC patients.Trail registration: Not applicable

Assessment of synovial fluid and serum cytokine levels in children with septic arthritis

Acute septic arthritis (ASA) is a common orthopedic infection of children which may produce devastating sequelae and chronic morbidity. Improved understanding of the intra-articular inflammatory response in ASA may identify cytokine targets with diagnostic or therapeutic potential, though no detailed investigations to this end have been performed. Given this, we used a multiplex cytokine assay for assessment of levels of 40 different cytokines in the synovial fluid and blood of children with ASA. Twelve children (8 controls undergoing orthopedic surgery for non-infectious conditions and 4 with ASA) were prospectively enrolled. Blood and synovial fluid were collected intraoperatively from each subject, and the levels of 40 cytokines were determined using a multiplex assay. Cytokines were organized by function and structure into 12 groups for analysis. The Benjamini-Hochberg method was used to control for type 1 errors, with an a priori false discovery rate of 10%. Subjects with ASA were younger than controls (mean age 8.0 vs 13.1 years, p=0.0400). Significant elevations were seen in interleukins (IL) with chemokine properties, IL-6 and those in the common-γ chain group in the blood and synovial fluid of children with ASA compared with controls, while significant elevations in 5 additional cytokine groups were seen in synovial fluid from children with ASA compared with controls, most notably IL-6 (median 8294.3 vs 10.7 pg/mL, p=0.0066). Our pilot study is the first to describe in detail the cytokine response in children with ASA, and highlights the need for additional study.

Data-driven analysis of COVID-19 reveals specific severity patterns distinct from the temporal immune response

Key immune signatures of SARS-CoV-2 infection may associate with either adverse immune reactions (severity) or simply an ongoing anti-viral response (temporality); how immune signatures contribute to severe manifestations and/or temporal progression of disease and whether longer disease duration correlates with severity remain unknown. Patient blood was comprehensively immunophenotyped via mass cytometry and multiplex cytokine arrays, leading to the identification of 327 basic subsets that were further stratified into more than 5000 immunotypes and correlated with 28 plasma cytokines. Low-density neutrophil abundance was closely correlated with hepatocyte growth factor levels, which in turn correlated with disease severity. Deep analysis also revealed additional players, namely conventional type 2 dendritic cells, natural killer T cells, plasmablasts and CD16+ monocytes, that can influence COVID-19 severity independent of temporal progression. Herein, we provide interactive network analysis and data visualization tools to facilitate data mining and hypothesis generation for elucidating COVID-19 pathogenesis.

Measurable genomic changes in Mycobacterium avium subsp. hominissuis after long-term adaptation in Acanthamoeba lenticulata and reduced persistence in macrophages

Free-living amoebae are ubiquitous in aquatic environments and act as environmental reservoirs for nontuberculous mycobacteria. Mycobacterium avium subsp. hominissuis recovered from Acanthamoeba has been demonstrated to be more virulent in both human and murine models. Here, we investigate the persistence of M. avium subsp. hominissuis after short-term (2 weeks) and long-term (42 weeks) co-culture in Acanthamoeba lenticulata. We hypothesize that A. lenticulata-adapted M. avium subsp. hominissuis demonstrate phenotypic and genomic changes facilitating intracellular persistence in naïve Acanthamoeba and human macrophages. M. avium subsp. hominissuis CFU in co-culture with A. lenticulata were recorded every 2 weeks up to 60 weeks. While A. lenticulata-associated M. avium subsp. hominissuis CFU did not significantly change across 60 weeks of co-culture, longer adaptation time in amoebae reduced colony size. Isolates recovered after 2 or 42 weeks of amoebae co-culture were referred as "early-adapted" and "late-adapted" M. avium subsp. hominissuis, respectively. Whole genome sequencing was performed on amoebae-adapted isolates with pan-genome comparisons to the original M. avium subsp. hominissuis isolate. Next, amoebae-adapted isolates were assessed for their persistence in A. lenticulata, A. castellanii, and human THP-1 macrophages. Multiplex cytokine/chemokine analyses were conducted on THP-1 culture supernatants. Compared to the original isolate, counts of late-adapted M. avium subsp. hominissuis were reduced in Acanthamoeba and contrary to expectations, lower counts were also observed in THP-1 macrophages with concomitant decrease in TNFa, IL-6, and MIP-1b suggesting that host adaptation may influence the inflammatory properties of M. avium. IMPORTANCE Short-term interaction between Acanthamoeba and M. avium has been demonstrated to increase infectivity in human and murine models of infection, establishing the paradigm that amoebae "train" M. avium in the environment by selecting for phenotypes capable of enduring in human cells. We investigate this phenomenon further by determining the consequence of long-term amoebae adaptation on M. avium subsp. hominissuis persistence in host cells. We monitored genomic changes across long-term Acanthamoeba co-culture and report significant changes to the M. avium subsp. hominissuis genome in response to amoebae-adaptation and reduced colony size. Furthermore, we examined isolates co-cultured with A. lenticulata for 2 or 42 weeks and provide biological evidence that long-term co-culture in amoebae reduces M. avium persistence in human macrophages.