Critical role of aldehydes in cigarette smoke-induced acute airway inflammation

Background

Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation.

Methods

BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs.

Results

Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein.

Conclusion

Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.     

Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.     

How citrullination invaded rheumatoid arthritis research

Citrullination and the immune response to citrullinated proteins have been fundamental for the early recognition of rheumatoid arthritis by serological tests and a better understanding of its pathophysiology. In the first years after the initial publications, the focus was on the antibodies directed to citrullinated proteins. It is now realized that citrullinating enzymes and citrullinated proteins may have important roles in the maintenance of the inflammatory processes in the joints. There is also accumulating evidence for a direct role of citrullination in tissue destruction in the rheumatoid synovium. Here we will discuss the development and importance of anti-citrullinated protein antibodies in rheumatoid arthritis as well as recent findings implicating citrullination in the pathophysiology of rheumatoid arthritis.The first indication that patients with rheumatoid arthritis (RA) produce antibodies to a specific autoantigen was published in 1964 by two Dutch scientists, Nienhuis and Mandema. The exact nature of this antigen, the so-called perinuclear factor, remained unclear for decades. In 1978, the target of seemingly unrelated RA-specific autoantibodies (that is, keratin) was identified. Almost 15 years later, Guy Serre’s group convincingly showed that both antigens were identical to the cytokeratin filament-aggregating protein filaggrin (reviewed in [1]). Our own previously published results had shown that the newly made precursor of filaggrin in cultured buccal mucosa cells (that is, profilaggrin) did not react with RA antibodies [2]. This prompted us to consider the possibility that a post-translational modification of filaggrin, absent on newly made profilaggrin, was required for the formation of the antigenic target of these antibodies. Since 1994, we have tested several likely modifications using synthetic peptides. Indeed, citrullination, the enzymatic conversion of peptidylarginine into peptidylcitrulline, turned out to be essential to make peptides reactive with RA autoantibodies. We subsequently developed an enzyme-linked immunosorbent assay with citrullinated peptides and confirmed that the anti-peptidylcitrulline activity was specific for RA [3]. Our further work was directed to the development of the CCP2 test, using cyclic citrullinated peptides (CCPs) selected from random peptide libraries [4].The discovery of CCP/protein as the most prominent RA-specific antigen had great impact on RA diagnostics and our understanding of RA pathophysiology. The following milestones can be noted (see [5] also).1. After decades of intensive research by many groups, a specific diagnostic test for RA had finally been developed. The CCP2 test has a specificity of more than 95%, is very sensitive (~75%), and is still considered the gold standard in RA autoantibody testing. Since 2010, anti-citrullinated protein antibodies (ACPAs) have been included in the new American College of Rheumatology/European League Against Rheumatism classification criteria for RA.2. Recently, an international reference preparation for ACPAs was evaluated by the International Committee for the Standardization of Autoantibodies in Rheumatic and Related Diseases [6]. It is available for the scientific community via the Centers for Disease Control and Prevention (Atlanta, GA, USA).3. A positive CCP2 test predicts the development of RA, often years before clinical confirmation (reviewed in [5]). It appears that time to RA diagnosis is shorter in patients with high anti-CCP2 titers at enrollment as compared with those with low titers [7].4. ACPA-positive RA is characterized by a more severe disease course. Early treatment of ACPA-positive individuals appears to be very effective.5. ACPA-negative patients (about 25% of the total RA population) generally display a much milder course of disease. About 35% of such ACPA-negative patients produce anti-carbamylated protein antibodies. Interestingly, the chemical product of carbamylation (that is, lysine converted to homocitrulline) is structurally very similar to citrulline [8].6. Specific human leukocyte antigen (HLA) genes (DRB1 shared epitope (SE) alleles) not only are the most important genetic risk factor for RA but also are strongly associated with the production of ACPAs.7. The best known environmental risk factor for RA, cigarette smoking, is a risk factor only for ACPA-positive and not for ACPA-negative RA [9]. There is increasing evidence that smoking acts as a trigger for anti-citrulline immunity and does so mainly in the context of certain HLA genes and certain other genetic risk factors.8. ACPAs and citrullinated antigens form immune complexes which stimulate the inflammatory process. Continuous production of such immune complexes ultimately results in the chronic inflammation, characteristic for RA (Figure 1).Open in a separate windowFigure 1Citrullination-related immunity and pathophysiology in rheumatoid arthritis. In genetically susceptible individuals, an environmental factor may initiate a primary inflammation, which can occur in various tissues, and trigger the immune response to citrullinated proteins (left). The resulting anti-citrullinated protein/peptide antibodies (ACPAs) are distributed through the circulation and may form immune complexes with citrullinated proteins produced in an inflamed synovium, thereby boosting the inflammatory process. This will be associated with the infiltration and activation of neutrophils, macrophages, and lymphocytes; cell death; extracellular DNA trap formation; the activation and release of peptidylarginine deiminases (PADs); de novo citrullination; and diversification of the ACPA response. Besides the common inflammation-associated mediators of tissue destruction (not shown), ACPAs and PADs can be directly involved in these processes. HLA, human leukocyte antigen.     

A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins

We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G > T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G > A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.     

Presence of c-kit positive cells in fetal and adult bovine forestomachs

The interstitial cells of Cajal (ICC) have been reported to regulate gastrointestinal motility. We investigated the distribution and the morphological and morphometric characteristics of the immunohistochemical reaction against c-kit in the forestomachs of fetal, newborn and adult cows. The anti-c-kit reaction revealed different populations of ICC among age groups and organs. ICC were more numerous and smaller in fetuses. Larger ICC were identified in newborns, except for those in the rumen. During the earliest stages of development, ICC were abundant in the inner layer of the muscularis and were consistently associated with this layer. In all samples, ICC were found in the outer layer of the tunica muscularis. ICC were found between the two muscle layers in the omasum at all ages; however, they were identified only in the rumen of the adult. Our study demonstrated that ICC are present in the forestomach of bovines.     

C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity

The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.     

Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway

The Notch pathway contributes to self-renewal of tumor-initiating cell and inhibition of normal colonic epithelial cell differentiation. Deregulated expression of Notch1 and Jagged1 is observed in colorectal cancer. Hairy/enhancer of split (HES) family, the most characterized targets of Notch, involved in the development of many cancers. In this study, we explored the role of Hes1 in the tumorigenesis of colorectal cancer. Knocking down Hes1 induced CRC cell senescence and decreased the invasion ability, whereas over-expression of Hes1 increased STAT3 phosphorylation activity and up-regulated MMP14 protein level. We further explored the expression of Hes1 in human colorectal cancer and found high Hes1 mRNA expression is associated with poor prognosis in CRC patients. These findings suggest that Hes1 regulates the invasion ability through the STAT3-MMP14 pathway in CRC cells and high Hes1 expression is a predictor of poor prognosis of CRC.