Therapeutic potential of targeting IL‐17 and IL‐23 in sepsis

Severe sepsis is a major concern of public health in industrialized countries. It is estimated that in the United States 200,000‐400,000 cases occur annually and resulting in an extensive burden for the health care systems. To date, no FDA‐approved pharmacologic agents for the treatment or prevention of human sepsis are available. The current modalities of therapy in sepsis include the standard arsenal of supportive interventions in critical care medicine and pharmacotherapy, with use of antibiotics and catecholamines. Despite such efforts, the mortality rates of sepsis have remained around 30‐50 %. Extensive scientific studies have utilized animal models of disease and aimed for a better understanding of the pathophysiologic mechanisms during sepsis. Members of the IL‐17 family of cytokines, as well as the functionally related IL‐23, have been identified as new players in the molecular events during sepsis. Strategies for targeting these mediators with neutralizing antibodies during experimental sepsis in rodents have demonstrated efficacy, resulting in improved survival outcomes. Currently, it is not clear whether such findings can be translated to human sepsis. This review highlights the current knowledge on the biology of IL‐17 isoforms and IL‐23 as well as potential applications to clinical medicine.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155453/1/ctm22001132614.pd

Modulation of inflammation by interleukin‐27

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141977/1/jlb1159.pd

Untersuchungen über die Regulation der Genexpression von Interleukin-18-Bindungsprotein und Interleukin-18

Interleukin-18-Bindungsprotein (IL-18BP) ist ein erst kürzlich entdeckter Gegenspieler von Interleukin-18 (IL-18). Aufgrund der Eigenschaft von IL-18BP mit hoher Affinität an IL-18 zu binden, wird IL-18 neutralisiert und seine biologischen Wirkungen durch IL-18BP inhibiert. Das Zytokin IL-18 ist ein multifunktioneller Botenstoff des Immunsystems, dessen Aktivität bei der Entstehung von Entzündungen, der Abwehr von Infektionen und der Rückbildung von Tumoren beteiligt sein kann. Eine der bedeutendsten Wirkungen von IL-18 ist insbesondere seine Fähigkeit die Produktion und Freisetzung von Interferon-gamma durch T-Helfer Typ 1 (Th1) Zellen, Natürliche Killer (NK) Zellen und CD8+ zytotoxische Zellen auszulösen. Bislang war lediglich bekannt, dass es sich bei IL-18BP um ein konstitutiv exprimiertes und sezerniertes Protein handelt. Die Zielsetzung dieser Promotionsarbeit war es zu untersuchen, ob eine Regulation der Genexpression von IL-18BP in Nicht-Immunzellen stattfindet. Dazu wurde im ersten Schritt eine semiquantitative RT-PCR Methode etabliert, mit Hilfe derer eine schwache konstitutive Expression der IL-18BP mRNA in Zellkulturen von humanen renalen Mesangiumzellen, epithelialen DLD-1 Kolonkarzinomzellen und Fibroblasten nachgewiesen wurde. Im Folgenden konnte als wesentliches Ergebnis festgestellt werden, dass eine Induktion der Genexpression von IL-18BP durch Interferon-gamma erfolgt. Mit RNase Protection Assays wurden nach Interferon-gamma Exposition 20 – 30fache relative Steigerungen der IL-18BP mRNA detektiert. In humanen Mesangiumzellen führte außerdem bakterielles Lipopolysaccharid zum Anstieg der IL-18BP Genexpression. Im zweiten Teil der Untersuchungen ließ sich unter Verwendung eines eigens hergestellten polyklonalen Antiserums nachweisen, dass durch Interferon-gamma auch eine starke Vervielfachung der Freisetzung bzw. Sekretion von IL-18BP stattfindet. Weiterhin wurden Kokulturen von IL-12/IL18 aktivierten humanen mononukleären Zellen aus dem peripheren Blut (PBMCs) mit entweder Mesangiumzellen oder DLD-1 Zellen durchgeführt. In diesen Kokulturen bewirkte die mittels ELISA gemessene Freisetzung von endogenem Interferon-gamma durch die PBMCs ebenfalls eine Induktion der Genexpression von IL-18BP in den Mesangiumzellen und DLD-1 Zellen. Darüber hinaus wurde in anderen Experimenten untersucht, ob die Regulation von IL-18BP gleichzeitig von Änderungen im Gehalt an IL-18 begleitet wird. Während in den humanen Mesangiumzellen kein IL-18 exprimiert wurde, konnte in den DLD-1 Zellen konstitutives proIL-18 detektiert werden. Jedoch hatte Interferon-gamma in DLD-1 Zellen keinen Einfluss auf die IL-18 Expression. Die hier zusammengetragenen Resultate belegen zum ersten Mal, dass es sich bei IL-18BP nicht nur um ein konstitutiv exprimiertes Protein, sondern vielmehr um einen spezifisch regulierten Immunmodulator handelt. Die Induktion der Freisetzung von IL-18BP durch Interferon-gamma stellt den entscheidenden Schritt eines bislang unbekannten negativen Rückkopplungsmechanismus zwischen Immunzellen und ortsständigen Nicht-Immunzellen dar: Nach der Freisetzung von IL-18 bei Entzündungen, Infektionen und Tumorerkrankungen führt das von Th1-, NK- und CD8+-Zellen produzierte Interferon-gamma zu einer Sekretion von IL-18BP durch Nicht-Immunzellen. Infolgedessen kommt es konsekutiv zur Limitierung der Aktivität von IL-18 mit Reduzierung seiner proinflammatorischen Wirkungen. Da ein Übermaß an IL-18 bei der Pathogenese von chronisch entzündlichen Erkrankungen wie beispielsweise der Rheumatoiden Arthritis und dem M. Chron eine Rolle zu spielen scheint, ist von besonderem Interesse welche natürlichen Wege für die Blockierung von IL-18 existieren. Die therapeutische Applikation von IL-18BP könnte sich in Zukunft als eine neue Strategie zur erfolgreichen Behandlung dieser Krankheiten erweisen.Interleukin-18 Binding Protein (IL-18BP) is a newly described opponent of Interleukin-18 (IL-18) which neutralizes IL-18 and inhibits its biological functions by binding IL-18 with high affinity. As a pleiotropic cytokine of the immune system, IL-18 has been shown to contribute to the pathogenesis of inflammation, the protective host defense against infection as well as tumor regression. One of the major actions of IL-18 is the ability to induce the production and release of Interferon-gamma from T helper type 1 (Th1) cells, Natural Killer (NK) cells and CD8+ cytotoxic cells. IL-18BP has previously been described to be constitutively expressed and secreted. This study focuses on the regulation of IL-18BP gene expression in non-leukocytic cells. A semiquantitative RT-PCR method was established and minute constitutive levels of IL-18BP mRNA were detected in cultures of human renal mesangial cells, the colon carcinoma cell line DLD-1 and fibroblasts. More important, IL-18BP mRNA expression was strongly upregulated by Interferon-gamma. Using RNase Protection Assays 20 – 30fold relative inductions of IL-18BP mRNA levels following Interferon-gamma stimulation were observed. In cultures of human mesangial cells Lipopolysaccharide also increased IL-18BP expression. Furthermore, experiments with a new polyclonal antiserum against IL-18BP revealed that Interferon-gamma also mediates release of IL-18BP into cell culture supernatants. In addition, expression of IL-18BP was studied in cocultures of human peripheral blood mononuclear cells (PBMCs) with either human mesangial cells or DLD-1 cells. Activation of PBMCs by the combination of IL-12/IL-18 resulted in the production of endogeneous Interferon-gamma detected by ELISA and was followed by the induction of IL-18BP gene expression in mesangial cells and DLD-1 cells. Finally, in separate experiments it was excluded that the regulation of IL-18BP is accompanied by changes in IL-18 expression. Whereas no expression of IL-18 was found in human mesangial cells, proIL-18 could be detected constitutively in DLD-1 cells. However, in DLD-1 cells Interferon-gamma did not regulate IL-18 expression. This study demonstrates for the first time that IL-18BP is not only constitutively expressed but rather represents a specific regulated modulator of the immune system. Induction of the release of IL-18BP by Interferon-gamma seems to be part of a new negative feedback mechanism linking functions of immune cells and non-leukocytic cells: Release of active IL-18 during inflammation, infection and cancer is followed by production of Interferon-gamma by Th1-, NK- and CD8+-cells. Subsequently, Interferon-gamma induces secretion of IL-18BP from non-leukocytic cells, thereby limiting further IL-18 activity. Since IL-18 seems to be implicated in the pathogenesis of chronic inflammatory diseases like rheumatoid arthritis or Chron´s disease, understanding of the natural mechanisms for neutralizing this cytokine might prove to be crucial. In future, administration of IL-18BP might evolve as a new therapeutic strategy for the treatment of chronic inflammatory diseases

Experimental design of complement component 5a‐induced acute lung injury (C5a‐ALI): a role of CC‐chemokine receptor type 5 during immune activation by anaphylatoxin

Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement‐derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6‐G+CD11b+ leukocytes to the alveolar spaces and severe alveolar‐capillary barrier dysfunction (C5a‐ALI; EC50[C5a] = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5adesArg) did not induce alveolar inflammation. The severity of C5a‐ALI was aggravated in C5‐deficient mice. Depletion of Ly6‐G+ cells and use of C5aR1‐/‐ bone marrow chimeras suggested an essential role of C5aR1+ hematopoietic cells in C5a‐ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC‐chemokines. Mice with genetic deficiency of CC‐chemokine receptor (CCR) type 5, the common receptor of chemokine (C‐C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a‐ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.—Russkamp, N. F., Ruemmler, R., Roewe, J., Moore, B. B., Ward, P. A., Bosmann, M. Experimental design of complement component 5a‐induced acute lung injury (C5a‐ALI): a role of CC‐chemokine receptor type 5 during immune activation by anaphylatoxin. FASEB J. 29, 3762‐3772 (2015). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154372/1/fsb2029009014.pd

Anti‐inflammatory effects of β2 adrenergic receptor agonists in experimental acute lung injury

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154493/1/fsb2026005038.pd

MyD88‐dependent production of IL‐17F is modulated by the anaphylatoxin C5a via the Akt signaling pathway

The interleukin‐17 (IL‐17) family of cytokines plays important roles in innate immune defenses against bacterial and fungal pathogens. While much is known about IL‐17A, much less information is available about the IL‐17F isoform. Here, we investigated gene expression and release of IL‐17F and its regulation by the complement system. IL‐17F was produced in mouse peritoneal elicited macrophages after TLR4 activation by LPS, peaking after 12 h. This effect was completely dependent on the presence of the adaptor protein MyD88. The copresence of the complement activation product, C5a (EC50=10 nM), amplified IL‐17F production via the receptor C5aR. In vitro signaling studies indicated that LPS or C5a, or the combination, caused phosphorylation of Akt occurring at threonine 308 but not at serine 473. Treatment of macrophages with pharmacologic inhibitors of PI3K‐Akt greatly reduced production of IL‐17F as well as mRNA for IL‐17F. In endotoxemia, C5a levels peaked at 6 h, while IL‐17F levels peaked between 6‐12 h. Full in vivo production of IL‐17F during endotoxemia required C5a. A similar result was found in the cecal ligation and puncture sepsis model. These data suggest that maximal production of IL‐17F requires complement activation and presence of C5a.—Bosmann, M., Patel, V. R., Russkamp, N. F., Pache, F., Zetoune, F. S., Sarma, J. V., Ward, P. A. MyD88‐dependent production of IL‐17F is modulated by the anaphylatoxin C5a via the Akt signaling pathway. FASEB J. 25, 4222–4232 (2011). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154335/1/fsb2fj11191205.pd

Complementâ induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction

Polymicrobial sepsis in mice causes myocardial dysfunction after generation of the complement anaphylatoxin, complement component 5a (C5a). C5a interacts with its receptors on cardiomyocytes (CMs), resulting in redox imbalance and cardiac dysfunction that can be functionally measured and quantitated using Doppler echocardiography. In this report we have evaluated activation of MAPKs and Akt in CMs exposed to C5a in vitro and after cecal ligation and puncture (CLP) in vivo. In both cases, C5a in vitro caused activation (phosphorylation) of MAPKs and Akt in CMs, which required availability of both C5a receptors. Using immunofluorescence technology, activation of MAPKs and Akt occurred in left ventricular (LV) CMs, requiring both C5a receptors, C5aR1 and â 2. Use of a waterâ soluble p38 inhibitor curtailed activation in vivo of MAPKs and Akt in LV CMs as well as the appearance of cytokines and histones in plasma from CLP mice. When mouse macrophages were exposed in vitro to LPS, activation of MAPKs and Akt also occurred. The copresence of the p38 inhibitor blocked these activation responses. Finally, the presence of the p38 inhibitor in CLP mice reduced the development of cardiac dysfunction. These data suggest that polymicrobial sepsis causes cardiac dysfunction that appears to be linked to activation of MAPKs and Akt in heart.â Fattahi, F., Kalbitz, M., Malan, E. A., Abe, E., Jajou, L., Huberâ Lang, M. S., Bosmann, M., Russell, M. W., Zetoune, F. S., Ward, P. A. Complementâ induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction. FASEB J. 31, 4129â 4139 (2017). www.fasebj.orgâ Fattahi, Fatemeh, Kalbitz, Miriam, Malan, Elizabeth A., Abe, Elizabeth, Jajou, Lawrence, Huberâ Lang, Markus S., Bosmann, Markus, Russell, Mark W., Zetoune, Firas S., Ward, Peter A., Complementâ induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction. FASEB J. 31, 4129â 4139 (2017)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154261/1/fsb2fj201700140r.pd

Tyrosine kinase 2 promotes sepsis‐associated lethality by facilitating production of interleukin‐27

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141056/1/jlb0123-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141056/2/jlb0123.pd

Extracellular histones are essential effectors of C5aR‐ and C5L2‐mediated tissue damage and inflammation in acute lung injury

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154331/1/fsb2027012034.pd

Role of extracellular histones in the cardiomyopathy of sepsis

The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca2+]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nachtâ , LRRâ , and PYDâ domainsâ containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca2+]i, as wellas defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.â Kalbitz, M., Grailer, J. J., Fattahi, F., Jajou, L., Herron, T. J., Campbell, K. F., Zetoune, F. S., Bosmann, M., Sarma, J. V., Huberâ Lang, M., Gebhard, F., Loaiza, R., Valdivia, H. H., Jalife, J., Russell, M. W., Ward, P. A. Role of extracellular histones in the cardiomyopathy of sepsis. FASEB J. 29, 2185â 2193 (2015). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154273/1/fsb2fj14268730.pd