Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive. Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH. Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH. Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH. Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

Reconstitution of circulating IgG in µMT immunoglobulin-deficient mice (n=6) was performed similar to methods published for IgG reconstitution in human immunodeficient patients (3).

In vivo right ventricular systolic pressure (RVSP) assessment
Mice were anesthetized with isoflurane (induction for 2 minutes at 5% concentration and maintained at 2-3% for duration of assessment) mixed with room air or 100% O2 using a Surgivet Isotec 4 precision vaporizer (Smiths Medical, Minneapolis, MN). Once mice were sedated, they were placed supine while spontaneously breathing the isoflurane/air mix through a rodent nosecone. A 27-gauge needle attached to a fluid filled disposable pressure transducer (Hospira, Inc., Lake Forest, IL) was introduced percutaneously into the thorax via a subxiphoid approach. Right ventricular systolic pressure (RVSP) were verified in real time and recorded using a MP100 data acquisition system with AcqKnowledge software version 3.9.1-100M (Biopac Systems, Inc., Goleta, CA). Animals from chronic hypoxia exposure were kept in hypoxic conditions until immediately before RVSP measurement. After hemodynamic E3 measurements, blood was collected from the heart and mice were euthanized via bilateral thoracotomy.  DyLight-594 (anti-mouse or anti-rabbit IgG, depending on primary Abs used; purchased from Vector Laboratories). For double-labeling secondary Abs, appropriate species-specific biotinylated Abs were used, followed by Streptavidin conjugated to either Alexa-594 (red) or Alexa-488 (green). All reagents were used at dilutions recommended by the manufacturer.
IHC staining on human lung sections was performed via standard technique using reagents from Vector Laboratories, briefly: after deparaffinization, antigen retrieval (30 mins boiling in pressure cooker in citrate buffer, pH 6.0) and blocking steps (avidin, 15 mins, and biotin, 15 mins), primary biotinylated anti-C3d mouse mAbs (1:300, (1) were added for 1 hr at RT, followed by RTU horseradish peroxidase (30 mins at RT) and development in DAB (5 mins).

Quantification of IHC and IF staining
For IHC: Stained sections were Aperio-scanned (see above), randomized images were extracted from the scanned files and analyzed via Metamorph software. Graphs plotted wherein "Expression" represents percent threshold and "artificial units" (AU) represent the product area (pixel) time grey intensity (6).
For IF: Tissue sections with immunofluorescent staining were scanned on Leica-Aperio Versa 8 system at 20x magnification. The acquired images were then analyzed using the Aperio E5 ImageScope software v12.4.2 with the Area quantification FL v1 algorithm. The red fluorescence threshold was selected to eliminate the out-of-fluorescence background. The entire lung section area was selected for quantification and the red fluorescence was normalized to the total lung area and the resulting ratio is presented as "Expression" in Artificial Units (AU).

Quantative real-time PCR
RNA for quantitative RT-PCR from lung and liver tissue was isolated using QIAzol Lysis Gene expression fold change was calculated after normalization to Hprt1 using the D/D Ct method.

In situ hybridization
RNAscope in situ hybridization platform (probes, reagents, methodology) was used (ACDBio, Newark, CA). Briefly, frozen OCT-embedded mouse lung tissue sections (prepared as described above, sectioned at 10um thickness) were fixed in cold 10% buffered formalin, hybridized with CCL2/MCP1 or CSF2/GM-CSF antisense mouse-specific probes and processed according to the E6 manufacturer's instructions. The bound probes were detected using Fast Red kit and counterstained with 50% Gill's hematoxyllin. Both positive and negative control probes were run in parallel to confirm specificity of the target probes. Both fluorescent and bright-field images were acquired (since bound Fast Red can be visualized in both channels) using Zeiss microscope with AxioVision digital imaging system.

In vitro experiments
Human pulmonary fibroblasts, isolated from the adventitial layer of the main pulmonary artery of IPAH patients (n= 3, Suppl. Table 2