TLRs and Bcl-2 family proteins in neutrophils of oral cavity cancer patients.

Human neutrophils (PMNs), the cells engaged in the early phase of anti-tumor response, express TLR2 and TLR6 that can modulate the Bcl-2 family proteins, regulating the intrinsic apoptotic pathway in these cells. The expression of TLRs and Bcl-2 family is controlled by means of activating the transcriptional signaling pathways that involve the p38 MAP kinase. As previously described, PMNs from cancer patients exert accelerated apoptosis associated with decreased expression of anti-apoptotic Mcl-1 protein. In the present study we have been interested in establishing the involvement of TLR2 and TLR6, and p38 MAP kinase in the Mcl-1-modulated apoptosis in PMNs of oral cavity cancer patients. The expression of these proteins in neutrophils and autologous peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot, the intensity of apoptosis was estimated by flow cytometry, caspase-9 activity by colorimetric assay, and the cytochrome c concentration by ELISA. The simultaneous decreased expression of examined TLRs receptors and Mcl-1 protein, associated with the acceleration of PMNs apoptosis, suggests that this process in PMNs controlled by Mcl-1 is dependent on the TLR2 and TLR6 signalling. Impaired TLRs expression can lead to insufficient activation of p38PAPK, resulting in low expression of antiapoptotic Mcl-1 protein responsible for shortened lifespan of the examined PMNs

TLR4 ligation induces expression of APRIL molecule in human neutrophils — a preliminary study

In the present study we investigate the consequences of TLR4 activation by LPS for the synthesis of a proliferation-inducing ligand (APRIL) by human neutrophils (PMNs), and the possible role of the ERK1/2 kinases signaling pathway. In order to make a comparison, the same examinations were carried out on autologous peripheral blood mononuclear cells (PBMCs). The levels of mRNA for APRIL and TLR4 were measured using the real-time PCR method. Western blot analysis was used to assay the expressions of APRIL and ERK1/2 in cell lysates. We discovered an increased expression of APRIL accompanying the increased expression of TLR4 in the LPS-stimulated PMNs and PBMCs. Furthermore, stimulation with LPS triggered similar changes in phospho-ERK1/2 proteins expression in those cells. The present study suggests that LPS plays a role in TLR4-ligation in APRIL induction through ERK1/2 pathway activation in human neutrophils and mononuclear cells of peripheral blood. The association between TLR4 activation and APRIL expression in examined leukocytes might have important implications for the  mmune response of the host exposed to TLR4 ligands such as LPS

Regulation of NO production by MAPK dual-specificity phosphatases (DUSP) in human neutrophils exposed to N-nitrosodimethylamine

437-444One of the enzymes responsible for nitric oxide (NO) production in neutrophils is the inducible nitric oxide synthase (iNOS). Changes in its expression may result from the activation of different signaling pathways, including MAPK, which lead to activation of various genes, including DUSP genes. DUSP induce the negative feedback loop leading to MAPK deactivation through their phosphorylation. Our study assessed the role of DUSP1, DUSP10 and DUSP16 with the participation of MAPK in the iNOS-dependent NO production by neutrophils exposed to xenobiotic, N-nitrosodimethylamine (NDMA). The obtained results suggest that N-nitrosodimethylamine enhances the expression of all tested proteins (except DUSP10) in the cytoplasmic and nuclear fractions of neutrophils. The JNK pathway inhibition resulted in an extenuation of iNOS, phospho-p38 and DUSP10 expression in the cytoplasmic fraction and DUSP1 expression in the nuclear fraction of neutrophils. Inhibition of the p38 pathway led to a lower expression of iNOS, DUSP16 and DUSP10 in the cytoplasmic fraction. No changes in the phospho-JNK and DUSP1 expressions were observed. With the results of this study we can conclude that DUSP are positive regulators of MAP kinases in NDMA-induced signaling pathway which lead to modulation of iNOS-dependent NO production in human neutrophils

Powstawanie przeciwciał przeciwneutrofilowych (ANCA) w kontekście formowania zewnątrzkomórkowych sieci neutrofilowych (NETs)

Przeciwciała przeciwneutrofilowe – ANCA skierowane są przeciw składowym cytoplazmy granulocytów obojętnochłonnych, a ich obecność obserwowana jest m.in. u osób z zapaleniem małych naczyń. Przeciwciała te zostały wykryte prawie 40 lat temu, ale mechanizmy ich powstawiania, nie są dokładnie poznane. Antygeny docelowe dla przeciwciał ANCA, takie jak mieloperoksydaza (MPO) oraz proteinaza 3 (PR3), wchodzą w skład ziarnistości znajdujących się w cytoplazmie neutrofilów. Granulocyty obojętnochłonne, pełniąc istotną rolę w nieswoistej odporności organizmu, po aktywacji mogą wyrzucać zewnątrzkomórkowe pułapki NETs, które mają zdolność do unieruchomienia i unieszkodliwienia patogenów. Wyeksponowane w NETs antygeny MPO i PR3 mogą zostać zaprezentowane komórkom układu odpornościowego i uruchomić kaskadę tworzenia autoprzeciwciał ANCA, które z kolei przyczyniają się do zapętlenia procesów autoreaktywności neutrofilów. Celem pracy było wyjaśnienie potencjalnej roli NETs w procesach powstawania przeciwciał ANCA

Significance of NETs Formation in COVID-19

Severe contagious respiratory disease—COVID-19—caused by the SARS-CoV-2 coronavirus, can lead to fatal respiratory failure associated with an excessive inflammatory response. Infiltration and spread of SARS-CoV-2 are based on the interaction between the virus’ structural protein S and the cell’s receptor–angiotensin-converting enzyme 2 (ACE2), with the simultaneous involvement of human trans-membrane protease, serine 2 (TMPRSS2). Many scientific reports stress the importance of elevated recruitment and activity of neutrophils, which can form extracellular neutrophil traps (NETs) playing a significant role in the mechanism of combating pathogens, in the pathogenesis of COVID-19. Excessive generation of NETs during prolonged periods of inflammation predisposes for the occurrence of undesirable reactions including thromboembolic complications and damage to surrounding tissues and organs. Within the present manuscript, we draw attention to the impact of NET generation on the severe course of COVID-19 in patients with concurrent cardiovascular and metabolic diseases. Additionally, we indicate the necessity to explore not only the cellular but also the molecular bases of COVID-19 pathogenesis, which may aid the development of dedicated therapies meant to improve chances for the successful treatment of patients. We also present new directions of research into medications that display NETs formation regulatory properties as potential significant therapeutic strategies in the progress of COVID-19

Significance of NETs Formation in COVID-19

Severe contagious respiratory disease—COVID-19—caused by the SARS-CoV-2 coronavirus, can lead to fatal respiratory failure associated with an excessive inflammatory response. Infiltration and spread of SARS-CoV-2 are based on the interaction between the virus’ structural protein S and the cell’s receptor–angiotensin-converting enzyme 2 (ACE2), with the simultaneous involvement of human trans-membrane protease, serine 2 (TMPRSS2). Many scientific reports stress the importance of elevated recruitment and activity of neutrophils, which can form extracellular neutrophil traps (NETs) playing a significant role in the mechanism of combating pathogens, in the pathogenesis of COVID-19. Excessive generation of NETs during prolonged periods of inflammation predisposes for the occurrence of undesirable reactions including thromboembolic complications and damage to surrounding tissues and organs. Within the present manuscript, we draw attention to the impact of NET generation on the severe course of COVID-19 in patients with concurrent cardiovascular and metabolic diseases. Additionally, we indicate the necessity to explore not only the cellular but also the molecular bases of COVID-19 pathogenesis, which may aid the development of dedicated therapies meant to improve chances for the successful treatment of patients. We also present new directions of research into medications that display NETs formation regulatory properties as potential significant therapeutic strategies in the progress of COVID-19

TLR4 ligation induces expression of APRIL molecule in human neutrophils — a preliminary study

In the present study we investigate the consequences of TLR4 activation by LPS for the synthesis of a proliferation-inducing ligand (APRIL) by human neutrophils (PMNs), and the possible role of the ERK1/2 kinases signaling pathway. In order to make a comparison, the same examinations were carried out on autologous peripheral blood mononuclear cells (PBMCs). The levels of mRNA for APRIL and TLR4 were measured using the real-time PCR method. Western blot analysis was used to assay the expressions of APRIL and ERK1/2 in cell lysates. We discovered an increased expression of APRIL accompanying the increased expression of TLR4 in the LPS-stimulated PMNs and PBMCs. Furthermore, stimulation with LPS triggered similar changes in phospho-ERK1/2 proteins expression in those cells. The present study suggests that LPS plays a role in TLR4-ligation in APRIL induction through ERK1/2 pathway activation in human neutrophils and mononuclear cells of peripheral blood. The association between TLR4 activation and APRIL expression in examined leukocytes might have important implications for the  mmune response of the host exposed to TLR4 ligands such as LPS

Role of ERK1/2 Kinase in the expression of iNOS by NDMA in human neutrophils

73-80<span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="" lang="EN-US">Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of <span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="" lang="EN-GB">kinase regulated by extracellular signals<span style="font-size: 11.0pt;mso-bidi-font-size:10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="" lang="EN-US"> (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells. </span