Long Covid Vasculitis

This post was was started a few hours after listening to a webinar on “COVID-19 Year Two: Looking Back/Looking Forward” presented May 20, 2021 12:00 PM Eastern Time (US and Canada).

• Diana Berrent, founder of Survivor Corps
• Dr. Harlan Krumholz, cardiologist and scientist at Yale University and Yale New Haven Hospital and co-founder of Hugo Health
• Dr. Elizabeth (Liz) Newman, scientific analyst at Morrison & Foerster LLP
• Matthew Karlyn, life sciences transactions + licensing partner at Morrison & Foerster LLP

One interesting take home message was that Ms Berrent, the woman responsible for the Covid citizen scientist database, is that vasculitis in one of the main symptoms of Long Covid is a vascular disease. Indeed, Mayo Clinic lists these general symptoms of vaculitis: fever, headache, fatigue, weight loss, and general aches and pains. This site also listed organ specific symptoms. Pain after eating was listed as one of the symptoms of digestive tract vasculitis.

Diagnostic blood tests. “These tests look for signs of inflammation, such as a high level of C-reactive protein. A complete blood cell count can tell whether you have enough red blood cells. Blood tests that look for certain antibodies — such as the anti-neutrophil cytoplasmic antibody (ANCA) test — can help diagnose vasculitis.” ANCA antibody tests detect myeloperoxidase and preteinase 3. Given the impact of Long Covid on the liver, we might order a smooth muscle actin test that is frequently used to detect autoimmune hepatitis. According to MedLine, symptoms of autoimmune hepatitis include: fatigue, jaundice (a condition that causes your skin and eyes to turn yellow), abdominal pain, joint pain, nausea, skin rashes, loss of appetite, dark-colored urine.

Dermatan sulfate binds autoantigens

This post looks at an explanation of where auto immunity comes from. Dermatan sulfate, a polysaccharide found in tendons, blood vessels, and skin. A Harvard group has published some very academic peer reviewed studies suggesting a role of dermatan sulfate in developing auto immunity as well as the production of auto antibodies. [1,2] Dermatan sulfate binds a lot of stuff and may be found in larger amounts in regions such as the skin [3] that are prone to more obscure Long Covid symptoms. The binding of auto antibodies to an auto antigen suggests the formation of an immune complex that these Wikipedia authors claim to sometime resulting in vasculitis.

The same Harvard group extracted proteins from a pulmonary epithelial cell line and isolated those that could bind to a dermatan sulfate affinity column. [4].

#pep	gene	protein	1.0 M	0.5 M	Auto Ag ref
9	ACTA2	Actin, aortic smooth muscle	+		[56]
8	ACTB	Actin, cytoplasmic 1	+		[57]
5	ACTBL2	Beta-actin-like protein 2	+		[57]
20	ACTN1	Alpha-actinin-1		+	[58]
20	ACTN4	Alpha-actinin-4		+	[56]
4	ANXA2	Annexin A2		+	[67]
10	ANXA3	Annexin A3		+	[68]
5	ANXA4	Annexin IV		+	[69]
15	ANXA5	Annexin A5		+	[70]
8	ANXA6	Annexin VI		+	[71]
27	FLNA	Filamin-A	+		[98]
25	FLNB	Filamin-B		+	[3]
9	FLNC	Filamin-C	+		[99]
2	TPM2	Tropomyosin 1 alpha chain		+	[206]
4	TPM3	Tropomyosin beta chain		+
8	TPM4	Tropomyosin alpha-3 chain		+	[207]
3	TSN	Tropomyosin alpha-4 chain		+	[208]
5	TUBA4A	Tubulin alpha-1C	+		[209]
4	TUBB4B	Tubulin alpha-4A	+		[210]
2	TXNDC5	Tubulin beta-2C chain	+		[211]
11	VIM	Vinculin		+	[217]
5	YWHAB	Vimentin	+		[219]

The annexins get honorable mention as cytoskeletal proteins

• Annexin 2 may cross link membrane phospholipids with the actin cytoskeleton and has been reported as a Covid autoantigen.
• Annexin 3 may have anticoagulant properties.
• Annexin IV binds membrane phospholipids, promotes membrane fusion, and is involved in exocytosis.
• Annexin V is used as a marker of apoptosis, programed cell death. When cells die this way, phosphatidyl serine (PS) is exposed on the outer membrane.
• Annexin VI may regulate the release of calcium from intracellular stores. In the image below A VI is releasing calcium from the endoplasmic reticulum.

The interesting aspect of this work is that many of these proteins are part of cytoskeletal complexes. Some of these images were taken from a SlideShare All of these proteins are abundant in smooth muscle.

In vitro smooth muscle auto antibodies

Back in 2005 a group from the University of Wisconsin tested the hypothesis that vascular smooth muscle had something tored them with do with vasculitis. These investigators isolated murine vascular smooth muscle cells and cultured them with mouse “splenocytes.” Splenocytes are white blood cells from the spleen that may include antigen presenting cells, T cells, and B cells. Milenyibiotech estimates that splenocytes are 21-25% T cells, 40% B cells and small amounts of antigen presenting cells.

” Freshly isolated splenocytes from naïve mice were co-cultured for 6 days under sterile conditions with irradiated syngeneic SM cell monolayers in a 10:1 ratio of splenocytes to SM cells. Floating cells were then harvested from the adherent cell monolayer by mild pipetting, washed in sterile Hanks’ balanced salt solution, and resuspended at 4 × 10⁷ cells/ml shortly before being transferred to recipient mice. ” [5] Note that these cells were irradiated. We may tentatively assume that these cells are dead and releasing their contents of actin, myosin, filamin, vinculin, and so on. If the the matrix contains dermatan sulfate, we may hypothesize that these cytoskeleton proteins are binding to it.

The authors used flow cytometry to count immune cell specific markers. [5] They made mention of thed prevalence of CD138 (syndecan-1) molecules, a phenotype suggesting B cells committed to a plasmablast phenotype. [5] SM activated B cells were tagged with fluorescent probes and injected into recipient mice. The activated B cells were found in the spleen and lungs. [5] These mice developed vasculitis. [5] Sera from these mice contained antibodies that reacted with vascular smooth muscle. [5]

• 

JhD mice, no mature B cells

Mice deficient in mature B cells (JhD strain, on BALB/c background) were used, both as donors and as recipients in vasculitis induction, and in subsequent serum transfers. Splenocytes deficient in B cells failed to be activated by the irradiated smooth muscle cell monolayer. (not shown)

T cell receptors are required

Rag2, plays a role in generating the diversity of T cell receptors. antibody mediated vascular damage required T cells, since wt and JhD mice developed the disease after vasculitic wt serum transfer, whereas RAG2^−/− mice did not. 

Antigen presenting B Cells and rouge T cells

• After binding of antigen by Ig (a.k.a B cell receptor) on B cell, the antigen is internalized by receptors mediated endocytosis and processed within the endocyte pathway into peptide.
• A personal hypothesis is that the actin peptides that T cells are exposed to in the thymus just different because they were not digested as complexes with binding partners.
• Antigen binding also initiates signaling through the B cell receptor that induces the B-cells to up regulate many membrane proteins, including class II MHC molecules and co-stimulatory ligand B7.
• Increased expression of both of these membrane proteins enhance the ability of B-cell to function as an antigen presenting cell (APC) in TH cell activation.
• Once the TH cell recognizes a processed antigenic peptide displayed by a class II MHC molecule on the membrane of B-cells, the two cells interact to form a T-B conjugate.
• This structural adjustment facilitates the release of cytokines towards the antigen specific B-cells.

What could all of this mean?

Maybe there was something about the cytokine storm and destruction of not only vascular smooth muscle but perhaps also airway and gastrointestinal smooth muscle. The Baiu (2005) study [5] suggests that all that is needed is a layer of dead smooth muscle cells, mature B cells, and T helper cells to keep them going to generate vasculitis autoantibodies. Later work of the Wang group [1,2,4] demonstrate the ability of dermatan sulfate to activate B cells and coincidentally, bind smooth muscle proteins. That dermatan sulfate is also found in the skin [2] may explain some other bizarre Long Covid symptom. It is suggested that we need an auto antibody test that includes not just actin but also cytoskeletal proteins found in smooth muscle, epithelial cells, and basically the stuff like filamin, alpha-actinin, and vinculin the Wang group [4] found associated with dermatan sulfate. There are easy ways to isolate large amounts of these proteins from porcine smooth muscle.

References

1. Wang J.Y., Lee J., Yan M., Rho J., Roehrl M.H.A. (2011) Dermatan sulfate interacts with dead cells and regulates CD5⁺ B-cell fate: implication for a key role in autoimmunity. Am J Pathol. 2011;178:2168–2176. [PMC free article]
2. Rho J.H., Zhang W., Murali M., Roehrl M.H., Wang J.Y. Human proteins with affinity for dermatan sulfate have the propensity to become autoantigens. Am. J. Pathol. 2011;178:2177–2190. [PMC free article]
3. Janet M. Trowbridge, Richard L. Gallo (2002) Dermatan sulfate: new functions from an old glycosaminoglycan, Glycobiology, Volume 12, Issue 9, 1 September 2002, Pages 117R–125R free article
4. Wang, J. Y., Zhang, W., Roehrl, M. W., Roehrl, V. B., & Roehrl, M. H. (2021). An autoantigen profile of human A549 lung cells reveals viral and host etiologic molecular attributes of autoimmunity in COVID-19. Journal of autoimmunity, 120, 102644. PMC free article
5. Baiu, D. C., Barger, B., Sandor, M., Fabry, Z., & Hart, M. N. (2005). Autoantibodies to vascular smooth muscle are pathogenic for vasculitis. The American journal of pathology, 166(6), 1851–1860. PMC free article