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. 2018 Nov 16:28:14.
doi: 10.1186/s12610-018-0079-x. eCollection 2018.

Effects of lipopolysaccharide-induced inflammation on hypoxia and inflammatory gene expression pathways of the rat testis

Michael A Palladino  1 , Genevieve A Fasano  1 , Dharm Patel  1 , Christine Dugan  1 , Marie London  1
Affiliations

Effects of lipopolysaccharide-induced inflammation on hypoxia and inflammatory gene expression pathways of the rat testis

Michael A Palladino et al. Basic Clin Androl. .

Abstract
in English, French

Background: Bacterial infection and inflammation of the testis impairs fertility, yet an understanding of inflammatory responses of the testis is incomplete. We are interested in identifying gene pathways involved in the detection and clearance of infectious microbes in the male reproductive tract. In previous studies in our lab focused on hypoxia-responsive genes of the testis, preliminary experiments suggested that genes classically categorized as hypoxia genes are also activated during antimicrobial responses. The purpose of this study was to identify hypoxia and inflammatory gene pathways that contribute to antimicrobial protection of the testis and to consider possible cross-talk and interactions between these pathways. Inflammation was induced in Sprague-Dawley rats using P. aeruginosa or E. coli lipopolysaccharide (LPS). Levels of hypoxia-inducible factor-1 (HIF-1α) protein and nuclear factor kappa B (NF-κB) were measured, and hypoxia and inflammatory gene expression patterns in testis were analyzed by gene expression profiling using real-time quantitative PCR arrays.

Results: In LPS-treated rats, HIF-1α protein increased with no change in Hif-1α mRNA. Western Blot analysis also demonstrated no change in NF-κB and inhibitory NFKB alpha (IκBα) protein levels following LPS treatment. Five hypoxia pathway genes (Angptl4, Egr1, Ier3, Pai1, and Glut1), and 11 inflammatory pathway genes (Ccl12, Cc13, Cd14, Cxcl10, Icam1, Il10, Il1b, Il6, Nfkbia, Tlr2, Tnf) up-regulated after 3 h of inflammation. Angptl4, Ccl12, Cc13, Cd14, Egr1, Nfkbia, Tlr2, and Tnf remained elevated at 6 h. Six genes were up-regulated at 6 h only (Bhlhe40, C3, Jak2, Nlrp3, Slc11a1, Tlr1). One gene (Tlr5) was down-regulated after 3 h and no genes at 6 h. Electrophoretic mobility shift assay results suggest a decrease in NF-κB binding activity following LPS treatment.

Conclusions: Testicular HIF-1α is up-regulated following LPS-induced inflammation. In contrast to other tissues, in which HIF-1α is up-regulated through transcriptional activation via NF-κB, we conclude that HIF-1α in the testis is not up-regulated through an increase in Hif-1α mRNA or through NF-κB-dependent mechanisms. Hypoxia pathway genes and genes involved in Toll-like receptor (TLR) and cytokine-mediated signaling comprise major functional categories of up-regulated genes, demonstrating that both hypoxia and classic inflammatory pathways are involved in inflammatory responses of the testis.

Contexte: L’infection et l’inflammation bactériennes du testicule altèrent la fertilité; cependant la compréhension des réponses inflammatoires du testicule est. encore incomplète. Nous nous sommes intéressés à l’identification des voies des gènes impliqués dans la détection et l’élimination des microbes infectieux dans l’appareil reproductif masculin. Dans de précédentes études menées dans notre laboratoire, et centrées sur des gènes sensibles à l’hypoxie, les expérimentations préliminaires suggéraient que les gènes classiquement catégorisés comme gènes de l’hypoxie étaient aussi activés au cours des réponses antimicrobiennes. Le but de la présente étude était d’identifier les voies des gènes qui contribuaient à la protection antimicrobienne du testicule et d’examiner de potentiels intermodulations et interactions entre ces voies.L’inflammation a été induite chez des rats Sprague-Dawley en utilisant des lypopolysaccharides (LPS) de P. aeruginosaet d’E. coli. Les taux de protéine du facteur-1 inductible par l’hypoxie (HIF1- α) et du facteur nucléaire kappa B (NF- kB) ont été mesurés; les profils d’expression des gènes de l’hypoxie et de l’inflammation dans le testicule ont été analysés par profilage de l’expression génique par PCR quantitative en temps réel.

Résultats: Chez les rats traités par LPS, la protéine HIF-1 α a augmenté sans modification de Hif-1αmRNA. L’analyse par Western Blot a aussi montré l’absence de modifications des taux de NF-kB et de la protéine inhibitrice NFKB alpha (IkB α) après traitement. Cinq gènes de la voie hypoxie (Angptl4, Egr1, Ier3, Pai1,et Glut1), et 11 gènes de la voie inflammatoire (Ccl12, Cc13, Cd14, Cxcl10, Icam1, Il10, Il1b, Il6, Egr1, Nfkbia, Tlr2, et Tnf) ont été régulés à la hausse après 3 heures d’inflammation. Angptl4, Ccl12, Cc13, Cd14, Egr1, Nfkbia, Tlr2, et Tnfsont restés élevés à 6 heures. Six gènes n’ont ont été régulés à la hausse qu’à 6 heures (Bhlhe40, C3, Jak2, Nlrp3, Slc11a1, Tlr1). Un gène (Tlr5) a été régulé à la baisse après 3 heures et aucun gène à 6 heures. Les résultats du test de décalage de la mobilité électrophorétique suggèrent une baisse de l’activité de liaison de NF- kB après traitement par LPS.

Conclusions: HIF-1α testiculaire est. régulé à la hausse après inflammation induite par LPS. Contrairement à d’autres tissus, dans lesquels HIF-1α est. régulé à la hausse par activation transcriptionnelle via NF- kB, nous concluons que HIF-1α dans le testicule n’est. pas régulé à la hausse par une augmentation de Hif-1 αmRNA ou par des mécanismes NF-kB-dépendants. Les gènes de la voie hypoxie et les gènes impliqués dans le récepteur Toll-like (TLR) et dans la signalisation médiée par les cytokines comprennent des catégories fonctionnelles majeures de gènes régulés à la hausse, ce qui démontre qu’à la fois les voies de l’hypoxie et les voies classiques de l’inflammation sont impliquées dans les réponses inflammatoires du testicule.

Keywords: Hypoxia; Inflammation; Lipopolysaccharide; Orchitis; Pathogens.

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Conflict of interest statement

Animal research protocols were approved by the Monmouth University IACUC and conform to guidelines established in The Guide for the Care and Use of Laboratory Animals.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Serum testosterone levels decrease following LPS-induced inflammation. *Indicates statistically significant difference as compared to sham (ANOVA, P < 0.05; n = 3–5); **Indicates P < 0.001. P.a., Pseudomonas aeruginosa
Fig. 2
Fig. 2
HIF-1α protein levels increase following LPS-induced inflammation in rat testis. a Results of HIF-1α immunoblot analysis of testicular cytoplasmic proteins isolated from sham and LPS treated animals. COS-7 cell line protein extract was included as a positive control and ß-actin was included as an internal control. HIF-1α was detected at ~ 120 kDa. Shown is a 6 h sham as an example but time-matched shams (1, 3 and 6 h) were used for all experiments and for quantitation. Representative time points and results are shown in panel (a). b Histogram shows relative HIF-1α protein levels normalized to ß-actin and as compared to sham for all time points and treatment samples analyzed. *Indicates statistically significant difference as compared to sham (ANOVA, P < 0.05; n = 4–7). **Indicates P < 0.001
Fig. 3
Fig. 3
Phospho-NF-κB and IκB protein levels do not change following LPS-induced inflammation in rat testis. a Results of phospho-NF-κB and phospho-IκB immunoblot analysis of testicular cytoplasmic proteins isolated from sham and LPS treated animals. RAW cell line protein extract was included as a positive control and ß-actin was included as an internal control. Representative time points and results are shown in panel (a). Histograms show relative phospho-IκB (b) and phospho-NF-κB (c) protein levels normalized to ß-actin and as compared to sham for all time points and treatment samples analyzed. No statistically significant differences in phospho-IκB and phospho-NF-κB protein levels were observed between sham and LPS treated groups (ANOVA, P < 0.05; n = 3–5). STD, protein molecular weight standards
Fig. 4
Fig. 4
Electrophoretic mobility shift assay (EMSA) for NF-κB binding to the HIF-1α gene. a NF-κB binding to a consensus sequence from the HIF-1α gene in both cytoplasmic and nuclear protein extracts decreased following LPS treatment. b Relative DNA binding activity as compared to sham. *Indicates statistically significant difference as compared to sham (ANOVA, n = 3–5; P < 0.05). **Indicates P < 0.001
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