mCLCA3 Modulates IL-17 and CXCL-1 Induction and Leukocyte Recruitment in Murine Staphylococcus aureus Pneumonia

The human hCLCA1 and its murine ortholog mCLCA3 (calcium-activated chloride channel regulators) are exclusively expressed in mucus cells and linked to inflammatory airway diseases with increased mucus production, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Both proteins have a known impact on the mucus cell metaplasia trait in these diseases. However, growing evidence points towards an additional role in innate immune responses. In the current study, we analyzed Staphylococcus aureus pneumonia, an established model to study pulmonary innate immunity, in mCLCA3-deficient and wild-type mice, focusing on the cellular and cytokine-driven innate inflammatory response. We compared clinical signs, bacterial clearance, leukocyte immigration and cytokine responses in the bronchoalveolar compartment, as well as pulmonary vascular permeability, histopathology, mucus cell number and mRNA expression levels of selected genes (mClca1 to 7, Muc5ac, Muc5b, Muc2, Cxcl-1, Cxcl-2, Il-17). Deficiency of mCLCA3 resulted in decreased neutrophilic infiltration into the bronchoalveolar space during bacterial infection. Only the cytokines IL-17 and the murine CXCL-8 homolog CXCL-1 were decreased on mRNA and protein levels during bacterial infection in mCLCA3-deficient mice compared to wild-type controls. However, no differences in clinical outcome, histopathology or mucus cell metaplasia were observed. We did not find evidence for regulation of any other CLCA homolog that would putatively compensate for the lack of mCLCA3. In conclusion, mCLCA3 appears to modulate leukocyte response via IL-17 and murine CXCL-8 homologs in acute Staphylococcus aureus pneumonia which is well in line with the proposed function of hCLCA1 as a signaling molecule acting on alveolar macrophages

effect on the course of murine pneumococcal pneumonia

Die ambulant erworbene Pneumonie ist eine der häufigsten lebensbedrohlichen Infektionserkrankungen der heutigen Industrieländer und geht mit hoher Morbidität und Mortalität einher. S. pneumoniae ist der häufigste Erreger der AEP. Eine inadäquate Immunantwort, hervorgerufen durch akute oder chronische Primärerkrankungen, Langzeitbeatmung oder Sepsis-assoziierte Immunparalyse, erhöht das Risiko, an einer Pneumokokkenpneumonie zu erkranken. Spezifische Strategien zur Verbesserung der angeborenen Immunabwehr der Lunge fehlen jedoch weitgehend. In vivo Studien zeigten, dass die lokale Immunstimulation mit bakteriellen Bestandteilen oder spezifischen synthetischen TLR-Agonisten die pulmonale Immunabwehr verbessern und protektiv bei verschiedenen nachfolgenden Lungeninfektionen wirkt. Die pulmonale Immunstimulation vor einer Infektion stellt somit möglicherweise eine vielversprechende alternative Therapiestrategie dar. Der toll-like Rezeptor 2 (TLR-2) erkennt neben Lipoteichonsäure und Peptidoglykanen auch Lipoproteine und Lipopeptide und ist bei der Pneumokokkenpneumonie an der Induktion einer frühen Immunantwort beteiligt. Eine spezifische Stimulation des TLR-2 mit dem synthetischen MALP-2 bewirkte darüber hinaus die Freisetzung von proinflammatorischen Zytokinen in vitro und die lokale Rekrutierung von Entzündungszellen in vivo. Ziel der vorliegenden Dissertationsarbeit war es, den Einfluss einer TLR-2-spezifischen lokalen Immunstimulation mit dem synthetischen Lipopeptid MALP-2 auf die pulmonale Immunantwort, die Erregerelimination und den klinischen Verlauf bei der Pneumokokkenpneumonie im etablierten Mausmodell zu untersuchen. Dabei wurde zunächst die Auswirkung von MALP-2 auf das angeborene Immunsystem der Lunge analysiert. Dazu erfolgte in vivo die intratracheale Applikation von MALP-2 und nachfolgend die Untersuchung der lokalen Zytokinfreisetzung und der Rekrutierung von Entzündungszellen in den bronchoalveolären Raum. Zusätzlich wurde die Rolle des TLR-2 anhand von Wildtyp- und TLR-2-defizienten Mäusen in vivo sowie in Zellkultur in vitro eruiert. In weiteren experimentellen Untersuchungen wurden Tiere 24 h nach intratrachealer Applikation von MALP-2 mit S. pneumoniae infiziert. Anschließend erfolgte die Analyse der lokalen Entzündungsreaktion sowie des klinischen Verlaufes bei der murinen Pneumokokkenpneumonie. Beachtung fanden hierbei insbesondere die pulmonale Zytokinproduktion und Leukozytenrekrutierung in den Atemwegen sowie die Erregerelimination und die Überlebensraten im Verlauf der Pneumokokkenpneumonie. Die lokale Applikation von MALP-2 in die Lunge bewirkte in Abhängigkeit von TLR-2 die lokale Freisetzung von inflammatorischen Zytokinen und Chemokinen sowie die Rekrutierung von Leukozyten in den bronchoalveolären Raum. MALP-2 erhöhte darüber hinaus die Genexpression des TLR-2 in der Lunge in vivo und in humanen Alveolarepithelzellen in vitro sowohl auf mRNA- als auch auf Proteinebene. Die lokale Applikation von MALP-2 24 h vor der intranasalen Infektion mit S. pneumoniae verursachte eine erhöhte Freisetzung von CCL5 (RANTES) in Verbindung mit einer gesteigerten Rekrutierung von Leukozyten und einer verminderten Produktion des anti- inflammatorischen IL-10 in den bronchoalveolären Raum. Die Gesamtleukozytenzahl und das Differentialzellbild im Blut blieben jedoch unverändert. Klinisch zeigten MALP-2-vorbehandelte Tiere im Vergleich zu kontrollbehandelten infizierten Tieren höhere Überlebensraten sowie weniger Gewichtsverlust und Abfall der Körpertemperatur bei der Pneumokokkenpneumonie. MALP-2 bewirkte ferner eine Reduktion der Bakteriämie und verbesserte die Elimination von S. pneumoniae im Lungenparenchym. Zusammenfassend zeigen die Ergebnisse, dass die pulmonale Immunstimulation mit MALP-2 vor der Infektion mit S. pneumoniae die lokale Immunabwehr verbessert und die Überlebensrate der murinen Pneumokokkenpneumonie erhöht. Die präventive lokale Immunstimulation könnte somit für Patienten mit einem erhöhten Risiko, an einer Pneumokokkenpneumonie zu erkranken, eine vielversprechende pharmakologische Interventionsstrategie darstellen.Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide, and Streptococcus pneumoniae is the major causative agent of.CAP. The risk of pneumococcal pneumonia may be greatly increased in specific pathologic situations with impaired pulmonary host defense including acute or chronic primary diseases, long-term ventilation or sepsis-associated immune paralysis. However, specific strategies to strengthen the pulmonary host defense are rare. Pre-activation of the pulmonary immune system with bacterial components or specific synthetic TLR-agonists has been reported to improve local host defense and increase resistance to various experimental pulmonary infections. Thus, pulmonary immune stimulation prior to infection may be a promising alternative therapeutic strategy. Toll-like receptor-2 (TLR-2) recognizes microbial components including lipoteichoic acid, peptidoglycan as well as lipoproteins and lipopeptides. TLR-2 contributes to the induction of early immune responses in pneumococcal pneumonia. Further, TLR-2 specific stimulation with MALP-2 induced the release of proinflammatory cytokines in vivo and recruited leukocytes to the local site of stimulation in vivo. In the current study, the effect of local TLR-2 mediated immune stimulation with MALP-2 on the pulmonary host defense, the bacterial clearance and the course of pneumococcal pneumonia was investigated in a murine model of pneumococcal pneumonia. Mice were treated with intratracheal injections of MALP-2 and the pulmonary innate immune response was analysed including inflammatory cytokine release and leukocyte recruitment into the bronchoalveolar space. In addition, the role of TLR-2 was investigated in vivo using wildtype and TLR-2-deficient mice, as well as in vitro, human alveolare epithelial cells. Furthermore, mice were infected with S. pneumoniae 24 h after intratracheal MALP-2 application. The pulmonary immune response and the course of murine pneumococcal pneumonia after MALP-2 pretreatment were examined with respect to pulmonary cytokine production, leukocyte immigration, bacterial clearance and survival rates. In the presence of TLR-2, intratracheal MALP-2 application evoked inflammatory cytokine and chemokine release, resulting in leukocyte immigration into the bronchoalveolar space. MALP-2 increased TLR-2 expression levels at both mRNA and protein level in murine lungs in vivo and in human alveolar epithelial cells in vitro. Pulmonary pretreatment with MALP-2 24 h before intranasal pneumococcal infection resulted in increased levels of CCL5 (RANTES) associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in the bronchoalveolar lavage fluid. Blood leukocyte numbers and populations remained unchanged. Importantly, MALP-2-pretreated as compared to untreated mice showed increased survival, decreased loss of body weight as well as reduced hypothermia in pneumococcal pneumonia. MALP-2 also reduced bacteremia and improved pneumococcal clearance in lung parenchyma. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia. Thus, preventive pulmonary immunostimulation may provide a promising pharmacological strategy for high-risk patients to improve pneumococcal pneumonia outcome

Proteasome β5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia.

Immunoproteasomes, harboring the active site subunits β5i/LMP7, β1i/LMP2, and β2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in β5i/LMP7-/- mice. Our data show that β5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in β5i/LMP7-/- mice. The impaired bacterial elimination in β5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections

Data_Sheet_1_Peptidoglycan Recognition Protein 2 Regulates Neutrophil Recruitment Into the Lungs After Streptococcus pneumoniae Infection.pdf

<p>Peptidoglycan (PGN) recognition proteins (PGLYRPs) are a highly conserved group of host defense proteins in insects and mammals that sense bacterial cell wall PGN and act bactericidally or cleave PGN by amidase function. Streptococcus (S.) pneumoniae is one of the top five killers worldwide and causes, e.g., pneumonia, endocarditis, meningitis and sepsis. S. pneumoniae accounts for approximately 1.5–2 million deaths every year. The risk of antibiotic resistance and a general poor prognosis in young children and elderly people have led to the need for new treatment approaches. To the best of our knowledge, there is no report on the relevance of PGLYRP2 in lung infections. Therefore, we infected mice deficient for PGLYRP2 transnasally with S. pneumoniae and examined the innate immune response in comparison to WT animals. As expected, PGLYRP2-KO animals had to be sacrificed earlier than their WT counterparts, and this was due to higher bacteremia. The higher bacterial load in the PGLYRP2-KO mice was accomplished with lower amounts of proinflammatory cytokines in the lungs. This led to an abolished recruitment of neutrophils into the lungs, the spread of bacteria and the subsequent aggravated course of the disease and early mortality of the PGLYRP2-KO mice. These data suggest a substantial role of PGLYRP2 in the early defense against S. pneumoniae infection, and PGLYRP2 might also affect other infections in the lungs.</p

mCLCA3-deficiency led to reduced protein quantities in BALF during infection without altered pulmonary vascular permeability.

<p>(A) Total protein of BALF was examined at indicated time points by BCA-assay. (B) Mouse albumin (MA) of transnasally infected or uninfected mice was measured by ELISA in BALF and plasma. Pulmonary vascular permeability was calculated from the MA BALF/plasma ratio. Values are given as mean ± SEM (n = 8 each group). ^#p<0.05, ^##p<0.01 versus the PBS control group. *p<0.05 as indicated.</p

mCLCA3 had no impact on severity or expansion of lung inflammation.

<p>(A) 24 hours after infection, left lungs of <i>Staphylococcus aureus</i> infected mice were used for quantification of lung lesions by Cavalieri principle. (B) Percentages of estimated lung lesion volumes and total lung volumes of the left lungs were calculated. Values are given as mean ± SEM (n = 4 each group).</p

No genotype differences in lung inflammation, lesion distribution or bacterial loads were observed during infection.

<p>Mice were transnasally infected with 5×10⁷<i>Staphylococcus aureus</i> Newman and killed at indicated time points. (A) Macroscopic examination revealed deeply red consolidated areas in the infected lungs (arrowhead) in contrast to PBS controls which behaved virtually identical. (B) Subsequently, lungs were fixed, embedded in paraffin and stained with hematoxylin and eosin for histopathological analyses. (C) Evidence of bacteria was assessed by immunohistochemistry with anti-<i>Staphylococcus aureus</i> antibody. Uninfected animals (left panel) served as negative controls. <i>Brown</i>, 3,3′-diaminobenzidine; <i>blue</i>, hematoxylin counterstain. (D) The total lung area affected by inflammation and (E) a lung inflammation score were determined. Values are given as mean ± SEM (n = 4 each group). ^#p<0.05 versus the PBS control group. <i>Bar</i> (B), 100 µm. <i>Bar</i> (C), 20 µm.</p

Lung Scoring Parameters.

<p>Lung Scoring Parameters.</p