“A proteomic approach to altered innate and adaptive immunity in the pathogenesis of PAH” – A Synopsis

Below is a synopsis of A proteomic approach to altered innate and adaptive immunity in the pathogenesis of PAH, a talk by Marlene Rabinovitch (MD, Stanford) given at the Vera Moulton Wall Center for Pulmonary Vascular Disease (video below). It is one of my favorite talks so far in this series of lectures…

  • Hypothesis: there is an abnormal immune response (both innate and adaptive) affecting pulmonary arteries that is common in all forms of PAH

Altered adaptive immunity in PAH pathogenesis

  • What if antigens produced in lung are the site for autoantibody formation and immune complex deposition directly in the lung and in the perivascular area?
  • SAMHD1 is an antiviral nuclear protein that suppresses HIV replication, and this is part of an innate immune response
  • Using Western immunoblot analysis, SAMHD1 protein expression was found to be increased in IPAH lung
  • Via Immunohistochemistry analysis:
    1. In controls, SAMHD1 expression is present in some of the inflammatory cells situated in the periphery
    2. In IPAH, abundant expression of SAMHD1 was found in occluded lesions and in perivascular region
  • Via confocal immunohistochemistry (immunofluorescence microscopy):
    1. Abundant expression of SAMHD1 observed in nuclei of endothelial cells of occluded lesions
    2. Colocalization of SAMHD1 was found in CD11 cells (dendritic cells), and in CD68+ (monocyte/macrophages)
  • How is SAMHD1 induced?
    1. SAMHD1 is induced in pulmonary arterial endothelial cells by TNF-alpha
  • Since SAMHD1 is an antiviral protein, is increased SAMHD1 an innate immune response to a virus localized to the pulmonary artery tissue?
  • Analyzed the virome of the IPAH lung tissues and found not much difference between IPAH and controls, with the exception of amplified expression of endogenous retroviruses from the HERV-K family, specifically HERV-KII
  • Why is a retrovirus expressed in PAH?
  • In zebrafish that are prone to cancer due to genomic instability, endogeneous retroviruses are enhanced.
  • In patients with PAH, there is genomic instability due to BMPR2 mutations. This could lead to HERV-K2 amplifications. As a consequence of this, HERV-K2 can induce cytokines that are implicated in PAH, such as TNF-alpha, IL-6, and SAMHD1, in macrophages, endothelial cells, and tertiary lymphoid tissue.
  • In embryonic stem cells, there is transient amplification of HERV-K2 to protect the embryonic stem cells. But as they differentiate, the HERV-K2 amplification disappears. Thus, it appears that HERV-K2 (the message/protein) can be amplified and can be repressed, but the DNA is present either way. So, perhaps in PAH patients, amplification of the HERV-K2 mRNA from the DNA never really quiets down or is re-activated by inflammatory stimuli.
  • HERV-K does indeed induce IL-6 in PAECs, and TNF-alpha in PBMCs
  • In IPAH lungs, HERV-K can be seen being expressed in CD68+ Adventitial Macrophages in the perivascular region, and this is not seen in control lung tissue
  • HERV-K has also been implicated in autoimmunity and in the activation of B Cells
  • In tertiary lymph nodes in close proximity to occluded vessels, SAMHD1 antibodies were detected.
  • In summary: HERV-K2 activation leads to a) endothelial and inflammatory cell activation which leads to increased SAMHD1 and b) activation of B cells in tertiary lymph nodes to produce SAMHD1 antibodies. The interaction of SAMHD1 antibodies and SAMHD1 leads to the formation of immune complexes followed by complement activation which leads to chronic inflammation and PAH. What can fuel this whole system is reduced BMPR2 which 1) enhances cytokine production and 2) provides some genomic instability that could amplify HERV-K

Altered innate immunity in PAH

  • When cells are under fire or there is tissue damage, elastases are released by neutrophils
  • Regardless of PH etiology, circulating elastase levels are elevated in PAH. That is, they are elevated in all forms of PH. You also see naturally occurring inhibitors like elafin, which are usually produced in abundance when there is heightened elastase activity, are suppressed in all forms of PH.
  • Increased neutrophil elastase is linked to autoimmunity in diabetes.
  • Neutrophils are recruited to site of injury, and it produces elastase. And elastase can condition dendritic cells to activate Treg cells to come in to protect the tissue. But if there is heightened elastase, there is transdifferentiation of anti-inflammatory Treg cells to proinflammatory Th17 cells.
  • The good news is you can control elastase levels by using elafin. If you recognize an elafin deficit, you can add recombinant elafin (given in Europe in clinical trials)
  • Elafin suppresses PMN (polymorphonuclear cell) elastase, PMN adhesion, netosis, cytokines, and autoimmunity, and improves BMPR2 function.
  • An alternative to controlling elastase is using Treg therapy. Treg therapy suppresses PMN and Monocyte activation and autoimmunity, as well as increases BMPR2
  • Using SPADE analysis with Flow Cytometry Time of Flight techniques, it was found that the SDF1 receptor (or CD184, or CXCR4) is increased in IPAH circulating dendritic cells, T cells, and B cells. This increase in SDF1 could explain why some of these cells can be recruited to the lung, and specifically the perivascular region.
  • Elevated pSTAT1 activity across all PH groups (IPAH, Scleroderma, and Drug & Toxin) in CD4+ CD25hi T cells (which include Treg cells)
  • It was found that Treg transdifferentiation into to proinflammatory STAT1 positive cells is implicated in Rheumatoid Arthritis
  • What may be happening here is that you no longer have a protective Treg cell, but rather a cell that can be contributing to the disease… if it can get to the “disease” location
  • The CD4+ CD25hi T cells were found to have elevated levels of GM-CSF receptor across all PH patients (IPAH, Scleroderma, and Drug & Toxin). Furthermore, since there is increased GM-CSF in IPAH pulmonary arteries in the intima, media, and adventitia (normal control pulmonary arteries do not have elevated GM-CSF in any of these regions)… this allows for these proinflammatory pSTAT1 positive CD4+ CD25hi T cells to target and be recruited to the pulmonary arteries.
  • Loss of BMPR2 + TNF-alpha stimulation enhances GM-CSF pulmonary arterial endothelial cells. The enhancement was on the translation side as opposed to the transcription side (i.e. GM-CSF mRNA translated more into protein, as opposed to GM-CSF gene being transcribed more into mRNA). This is interesting because when cells are under stress/assault… they undergo a protective ER stress response where they produce stress granules that keep mRNA’s from being very actively translated. But what is happening here is abnormally increased translation of cytokines… increased GM-CSF + IL6 + IL8
  • Heightened GM-CSF causes recruitment of all cells that have the GM-CSF receptor… including monocytes and macrophages… as well as the aforementioned proinflammatory Treg cells.

Promising Therapies to Reverse Abnormal Innate and Adaptive Immunity:

  • Tacrolimus – activates BMPR2 and is immunosuppressive, and can low doses can increase and improve function of T reg cells
  • Elafin – a natural elastase inhibitor, activates BMPR2
  • Bestatin – a leukotriene B4 inhibitor
  • IL-6 Receptor Antagonist
  • T reg Immunotherapy
  • All these have shown positive results in pre-clinical studies


  • Amplification of HERVK occurs in PAH and can induce a chronic innate immune antiviral response leading to the deposition of SAMHD1 immune complexes which causes inflammation and…
  • …heightened elastase activity in PAH (reflecting the abnormal innate immunity) that could lead to abnormal adaptive immunity and autoimmunity by switching T regs to proinflammatory cells which are…
  • ….recruited to pulmonary arteries to induce PH due to heightened GM-CSF in pulmonary arteries that are caused by 1) BMPR2 mutations 2) heightened inflammation
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