To differentiate MCs and to study their functionality, atmospheric oxygen conditions are traditionally used in cell culture although the physiological oxygen level in vivo is much lower. However, the effect of physioxia (here defined as 7% O2) during MC differentiation in vitro and consequently the effect on the functionality as immune cells is unexplored. To adress this issue, the differentiation rate, transcript expression of hif-1α, selected target genes, foxo3 and vhl as well as ROS production and histamine content, as functional markers of MCs were evaluated.
30 4. Antimicrobial Activity of Mast Cells: Role and Relevance of Extracellular
DNA Traps
Manuscript adjusted to style of respective journal
Mini Review ARTICLE
Front. Immunol., 18 July 2016 | https://doi.org/10.3389/fimmu.2016.00265
Title: Antimicrobial Activity of Mast Cells: Role and Relevance of Extracellular DNA Traps
Authors: Helene Möllerherm1, Maren von Köckritz-Blickwede1,2*† and Katja Branitzki-Heinemann1*†
1Department of Physiological Chemistry, University for Veterinary Medicine Hannover, Hanover, Germany
2Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Hanover, Germany
*Correspondence:
Maren von Köckritz-Blickwede, maren.von.koeckritz-blickwede@tiho-hannover.de;
Katja Branitzki-Heinemann, katja.branitzki-heinemann@tiho-hannover.de
†Maren von Köckritz-Blickwede and Katja Branitzki-Heinemann contributed equally.
31 The extent of Helene Möllerherm´s contribution to the article is evaluated according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B 2. Performing of the experimental part of the study: B
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: B
32 Abstract
Mast cells (MCs) have been shown to release their nuclear DNA and subsequently form mast cell extracellular traps (MCETs) comparable to neutrophil extracellular traps, which are able to entrap and kill various microbes. The formation of extracellular traps is associated with the disruption of the nuclear membrane, which leads to mixing of nuclear compounds with granule components and causes the death of the cell, a process called ETosis. The question arises why do MCs release MCETs although they are very well known as multifunctional long-living sentinel cells? MCs are known to play a role during allergic reactions and certain parasitic infections. Nonetheless, they are also critical components of the early host innate immune response to bacterial and fungal pathogens: MCs contribute to the initiation of the early immune response by recruiting effector cells including neutrophils and macrophages by locally releasing inflammatory mediators, such as TNF-α. Moreover, various studies demonstrate that MCs are able to eliminate microbes through intracellular as well as extracellular antimicrobial mechanisms, including MCET formation similar to that of professional phagocytes. Recent literature leads to the suggestion that MCET formation is not the result of a passive release of DNA and granule proteins during cellular disintegration, but rather an active and controlled process in response to specific stimulation, which contributes to the innate host defense. This review will discuss the different known aspects of the antimicrobial activities of MCs with a special focus on MCETs, and their role and relevance during infection and inflammation.
33 5. Formation of Neutrophil Extracellular Traps under Low Oxygen Level
Manuscript adjusted to style of respective journal
Hypothesis & Theory ARTICLE
Front. Immunol., 25. November 2016 | https://doi.org/10.3389/fimmu.2016.00518
Title: Formation of Neutrophil Extracellular Traps under Low Oxygen Level
Authors: Katja Branitzki-Heinemann1*, Helene Möllerherm1*, Lena Völlger1*, Diab M.
Husein1, Nicole de Buhr1,2, Stefanie Blodkamp1, Friederike Reuner1, Graham Brogden1, Hassan Y. Naim1 and Maren von Köckritz-Blickwede1,2†
1Department of Physiological Chemistry, University for Veterinary Medicine Hannover, Hanover, Germany
2Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Hanover, Germany
*Katja Branitzki-Heinemann, Helene Möllerherm, and Lena Völlger have contributed equally.
†Correspondence: Maren von Köckritz-Blickwede, maren.von.koeckritz-blickwede@tiho-hannover.de
34 The extent of Helene Möllerherm´s contribution to the article is evaluated according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: A 2. Performing of the experimental part of the study: A
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: B
35 Abstract
Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or Staphylococcus (S.) aureus wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was significantly increased under hypoxia. Interestingly, NET formation in response to viable S. aureus wild-type or nuclease-deficient strain was retained under hypoxia. Our results lead to the conclusion that hypoxia is not the ideal tool to analyze HIF-1α in neutrophils. However, the data clearly suggest that neutrophils react differently under hypoxia compared to normoxia and thereby highlight the importance of the usage of physiological relevant oxygen level when studying neutrophil functions.
36 6. Hypoxia Modulates the Response of Mast Cells to Staphylococcus aureus
Infection
Manuscript adjusted to style of respective journal
Original Research ARTICLE
Front. Immunol., 11. May 2017 | https://doi.org/10.3389/fimmu.2017.00541
Title: Hypoxia Modulates the Response of Mast Cells to Staphylococcus aureus Infection
Authors: Helene Möllerherm1, Katja Branitzki-Heinemann1, Graham Brogden1, Ayssar A.
Elamin2, Wulf Oehlmann2, Herbert Fuhrmann3, Mahavir Singh2, Hassan Y. Naim1 and Maren von Köckritz-Blickwede1,4*
1Department of Physiological Chemistry, University for Veterinary Medicine Hannover, Hanover, Germany
2LIONEX Diagnostics & Therapeutics, Braunschweig, Germany
3Faculty of Veterinary Medicine, Institute of Biochemistry, University of Leipzig, Leipzig, Germany
4Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Hanover, Germany
*Correspondence: Maren von Köckritz-Blickwede, maren.von.koeckritz-blickwede@tiho-hannover.de
37 The extent of Helene Möllerherm´s contribution to the article is evaluated according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B 2. Performing of the experimental part of the study: C
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: B
38 Abstract
To study the antimicrobial function of immune cells ex vivo, cells are commonly cultivated under atmospheric oxygen concentrations (20–21%; normoxia), although the physiological oxygen conditions in vivo are significantly lower in most tissues. Especially during an acute infection, oxygen concentration locally decreases to hypoxic levels around or below 1%. The goal of this study was to investigate the effect of hypoxia on the activity of mast cells (MCs). MCs were cultivated for 3 or 24 h at 1% O2 in a hypoxia glove box and co-incubated with heat-inactivated Staphylococcus aureus. When incubating the cells for 24 h under hypoxia, the transcriptional regulator hypoxia-inducible factor 1α (HIF-1α) was stabilized and resulted in increased extracellular trap formation and decreased phagocytosis. Interestingly, while phagocytosis of fluorescent S. aureus bioparticles as well as the release of extracellular traps remained unaffected at 3 h hypoxia, the secretion of the prestored mediator histamine was increased under hypoxia alone. In contrast, the release of TNF-α was generally reduced at 3 h hypoxia.
Microarray transcriptome analysis revealed 13 genes that were significantly downregulated in MCs comparing 3 h hypoxia versus normoxia. One interesting candidate is sec24, a member of the pre-budding complex of coat protein complex II (COPII), which is responsible for the anterograde transport of proteins from the ER to the Golgi apparatus. These data lead to the suggestion that de novo synthesized proteins including crucial factors, which are involved in the response to an acute infection like TNF-α, may eventually be retained in the ER under hypoxia. Importantly, the expression of HIF-1α was not altered at 3 h. Thus, our data exhibit a HIF-1α-independent reaction of MCs to short-term hypoxia. We hypothesize that MCs respond to short-term low oxygen levels in a HIF-1α-independent manner by downregulating the release of proinflammatory cytokines like TNF-α, thereby avoiding uncontrolled degranulation, which could lead to excessive inflammation and severe tissue damage.
39 7. Differentiation and functionality of bone marrow-derived mast cells depend
on varying physiologic oxygen conditions
Manuscript adjusted to style of respective journal
Hypothesis & Theory ARTICLE
Front. Immunol. 14. November 2017 | doi: 10.3389/fimmu.2017.01665
Title: Differentiation and functionality of bone marrow-derived mast cells depend on varying physiologic oxygen conditions
Authors: Helene Möllerherm1, Karsten Meier1, Kathrin Schmies1, Herbert Fuhrmann2, Hassan Y. Naim1, Maren von Köckritz-Blickwede1,3*, Katja Branitzki-Heinemann1*
1Department of Physiological Chemistry, University for Veterinary Medicine Hannover, Hanover, Germany
2Faculty of Veterinary Medicine, Institute of Biochemistry, University of Leipzig, Leipzig, Germany
3Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Hanover, Germany
*Contributed equally
*Correspondence: Katja Branitzki-Heinemann, katja.branitzki-heinemann@tiho-hannover.de and Maren von Köckritz-Blickwede, maren.von.koeckritz-blickwede@tiho-hannover.de
40 The extent of Helene Möllerherm´s contribution to the article is evaluated according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B 2. Performing of the experimental part of the study: B
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: C
41 Abstract
Mast cells (MCs) are long-living multifunctional innate immune cells that originate from hematopoietic precursors and specifically differentiate in the destination tissue, e.g., skin, respiratory mucosa, intestine, where they mediate immune cell recruitment and antimicrobial defense. In vivo these tissues have characteristic physiological oxygen levels that are considerably lower than the atmospheric oxygen conditions (159 mmHg, 21% O2; 5% CO2) traditionally used to differentiate MCs and to study their functionality in vitro. Only little is known about the impact of physiological oxygen conditions on the differentiation process of MCs. This study aimed to characterize the differentiation of immature murine bone marrow-derived MCs under physioxia in vitro (7% O2; 53 mmHg;
5% CO2). Bone marrow-derived suspension cells were differentiated in the presence of interleukin-3 with continuous, non-invasive determination of the oxygen level using a Fibox4-PSt3 measurement system without technique-caused oxygen consumption.
Trypan blue staining confirmed cellular viability during the specified period. Interestingly, MCs cultivated at 7% O2 showed a significantly delayed differentiation rate defined by CD117-positive cells, analyzed by flow cytometry, and reached >95% CD117 positive population at day 32 after isolation. Importantly, MCs differentiated under physioxia displayed a decreased transcript expression level of hif-1α and selected target genes vegf, il-6, and tnf-α, but an increase of foxo3 and vhl expression compared to MCs cultivated under normoxia. Moreover, the production of reactive oxygen species as well as the amount of intracellular stored histamine was significantly lower in MCs differentiated under low oxygen levels, which might have consequences for their function such as immunomodulation of other immune cells. These results show for the first time that physioxia substantially affect maturation and the properties of MCs and highlight the need to study their function under physiologically relevant oxygen conditions.
42 8. Discussion
Taken together, all presented projects highlight the importance of physiological oxygen levels for in vitro cell culture systems when studying the function and differentiation of innate immune cells.
Innate immune cells provide the first line of defense against infections. Upon infection, these cells need to function fast and efficiently to fight against invading pathogens, although acute local tissue hypoxia is occuring due to overconsumption of oxygen by pathogens and recruited immune cells (Eltzschig et al., 2011). The adaptation to oxygen shortages is mediated by HIF-1α, the central regulator of the cell to oxygen stress (Zinkernagel et al., 2007). It has already be shown that this transcription factor has a high impact e.g. on the bactericidal activity of myeloid cells by enhancing their bactericidal capacity (Peyssonnaux et al., 2005). It was questioned in these present studies if hypoxia and consequently inevitably associated HIF-1α play a role in NET release in neutrophils as well as in the antimicrobial activity of MCs. Furthermore the differentiation process und the function of MCs under their physiological oxygen level were investigated.
Condensed, the overall goal of this study was to evaluate the effect of oxygen shortages on innate immune cell function against zoonotic bacterial infections. To reach this goal, the following aims were placed in focus:
The impact of hypoxia on neutrophil extracellular trap (NET) formation (Aim1, Chapter 5), the effect of oxygen shortage on MC functionality as response to a bacterial infection (Aim 2, Chapter 6) and the effect of physioxia on murine bone-marrow derived MC differentiation and function in vitro (Aim 3, Chapter 7).
It was already shown for MCs that pharmacological boosting of HIF-1α by Akebia-4924 results in augmentation of MCET release (Branitzki-Heinemann et al., 2013).
Furthermore, in neutrophils it could be proven by data of our own laboratory, that HIF-1α might be involved in the formation of NETs by pharmacological stabilization of HIF-1α by
43 DFO and DMOG (Völlger et al., 2016). Thereby, DFO and DMOG also enhanced the release of NETs in human and bovine neutrophils in a ROS-dependent manner (Völlger et al., 2016). Based on these results of boosting HIF-1α in MCs and neutrophils under normoxia, we hypothesized that NET and MCET formation as well as other antimicrobial activities, like mediator release and phagocytosis by MCs may be similarly increased under more physiological hypoxic conditions, since HIF-1α is a global regulator of the cellular response to low oxygen. In the present thesis, this result was confirmed by the exposure of MCs to long-term hypoxia (24 h at 1% oxygen), when HIF-1α is stabilized resulting in a HIF-1α-dependent mechanism of adaptation by increasing MCET release (Chapter 6, figure 2 and 3A; Möllerherm et al., 2017). Contrary to these previous results, we show here that both, neutrophils and MCs, may adapt to short-term hypoxia (2 h for neutrophils and 3 h for MCs) in a 1α-independent manner. We presented that HIF-1α might not be responsible for the altered NET-phenotype in neutrophils under hypoxia (Chapter 5, figure 2 and 3; Branitzki-Heinemann, Möllerherm, Völlger et al., 2016).
Nevertheless, these results are based on the transcript expression level in neutrophils, and further investigations on the protein level need to be conducted since HIF-1α is stabilized on the protein level under hypoxia (Branitzki-Heinemann, Möllerherm and Völlger et al., 2016). Since it is shown that pharmacological stabilization of HIF-1α results in increased MCET formation (Branitzki-Heinemann et al., 2013) and increased NET formation (Völlger et al., 2016), it could be critically discussed, if hypoxia is an ideal tool to study HIF-1α stabilization in MC and neutrophils in vitro. An additional or longer hypoxic trigger seems to be needed under natural occurring physiological oxygen conditions to induce the stabilization of HIF-1α as it is shown for the long-term hypoxic adaptation of MCs. Short-term hypoxia alone does not alter the expression level of hif-1α in neutrophils or the protein and expression level in MCs. Nevertheless, we were not able to reproduce the same effect like the pharmacological boosting of HIF-1α under hypoxia; in any case, the antimicrobial activity of MCs and neutrophils is altered under short-term hypoxia.
Interestingly, MCs modulate their main antimicrobial activity, the degranulation of mediators, under short-term hypoxia independently of HIF-1α, shown on protein and on transcript level, by increasing the histamine release and reducing TNF-α release
44 (Möllerherm et al., 2017). Especially the decreasing mediator release of MCs could have a significant effect on the recruitment of effector immune cells. This hypothesis is assisted through the fine-tune release of the vasoactive mediator histamine, an important endogenous modulator of inflammatory and immune responses, which is responsible for the vascular permeability to enhance the blood flow and to allow the recruitment of other effector cells, which was shown to be significantly increased under hypoxia, but not under normoxia, in response to S. aureus (Chapter 6, figure 5B;
Möllerherm et al., 2017). Furthermore, it was shown before that histamine inhibits the TNF-α release in a concentration and time-dependent manner (Bissonnette et al., 1996), leading to the suggestion that MCs act more immunomodulatory under hypoxia to reduce an overwhelming inflammation by recruited immune effector cells (Möllerherm et al., 2017). This theory is also supported by data from studying the differentiation of MCs under physioxia (7% O2; 53 mmHg; 5% CO2). Mature physioxic differentiated MC display a reduced ROS-production and reduced cellular histamine storage, which might also have important consequences for mediating immune cell recruitment and antimicrobial defense functions (Chapter 7, figure 4A and 5A, Möllerherm et al.
submitted). This downregulation of intracellular stored histamine through physiological differentiation and also released proinflammatory cytokines like TNF-α during acute hypoxic conditions could avoid uncontrolled degranulation, which could lead to excessive inflammation and severe tissue damage (Hoenderdos et al., 2016).
These results were the first evidence that MCs and neutrophils may adapt to short-term hypoxia, as found during the early phase/ acute phase of infection, in a HIF-1α independent manner. Furthermore, these findings lead to the suggestion that beside HIF-1α, other regulatory factors play a role in the early adaptation to hypoxic stress.
Beside HIF-1α also other HIF-isoforms, like HIF-2α and HIF-3α, are known for their role in oxygen adaptation (Tian et al., 1997). The comprehensive function of HIF-3α remains nearly unknown, but a negative regulatory function of HIF-1α and HIF-2α is discussed, by competing for available HIF-1β (Yamashita et al., 2008) or by dimerizing with HIF-1α (Makino et al., 2001). HIF-2α gained more attention in the recent years; it is expressed in many different cell types including cells of the innate and adaptive immune system
45 and promising an important role in the adaptation to oxygen shortages (Palazon et al., 2014). Whether HIF-1α or HIF-2α is stabilized or even both depends on various intrinsic and extrinsic factors, like hypoxia or the activation status of the cell, subsequently leading to the regulation of overlapping or distinct sets of target genes (Palazon et al., 2014). The stabilization and degradation procedure of HIF-2α is similar to HIF-1α, both dimerize with HIF-1β to transcribe target genes. HIF-2α is expressed in diverse forms but not ubiquitously, like HIF-1α. The expression is limited to a few cell types, like in endothelial cells (Hu et al., 2003) in macrophages in relation to tumors (Imtiyaz et al.,
45 and promising an important role in the adaptation to oxygen shortages (Palazon et al., 2014). Whether HIF-1α or HIF-2α is stabilized or even both depends on various intrinsic and extrinsic factors, like hypoxia or the activation status of the cell, subsequently leading to the regulation of overlapping or distinct sets of target genes (Palazon et al., 2014). The stabilization and degradation procedure of HIF-2α is similar to HIF-1α, both dimerize with HIF-1β to transcribe target genes. HIF-2α is expressed in diverse forms but not ubiquitously, like HIF-1α. The expression is limited to a few cell types, like in endothelial cells (Hu et al., 2003) in macrophages in relation to tumors (Imtiyaz et al.,