Complement C5a Receptors in Hidradenitis Suppurativa

Hidradenitis suppurativa is a debilitating chronic inflammatory follicular skin disease. The estimated prevalence of HS range between 1 and 4%, even though it has been an underdiagnosed disease until recently (Revuz J, 2009). In the acute stage, patients present with painful inflamed nodules (boils) and abscesses in the groin, buttocks and axillae. In a late stage sinus tract formation and scarring occurs. Treatment options include the antibiotics, anti-TNF and surgical methods (wide excisions and deroofing). However, most patients do not respond, or only respond partially or temporarily to treatments (von der Werth JM, 2001; Napolitano M, 2017; Nazary M, 2011; van der Zee HH, 2012).

The pathogenesis of HS is largely unknown, and possible causes include plugged apocrine gland or hair follicle, excessive sweating, androgen dysfunction and genetic disorders. Defective cytokine responses and the infiltration of a variety of immune cells suggest the autoinflammatory nature of the disease (Giamarellos-Bourboulis EJ, 2007; van der Zee HH; 2011; van der Zee HH, 2012), which is in line with the significant improvement seen in patients administered the tumor necrosis factor-a (TNF-a) blocker adalimumab (Kimball AB, 2016). Neutrophils have been identified in skin lesions and are considered to be the major cell type to produce pus. Complement C5a is a major chemotactic factor that stimulates neutrophil infiltration. The role of C5a has just recently started to be studied in HS. (Blok JL, 2016; Kanni T, 2018). An open label phase 2 study demonstrated that a 50% HS clinical response rate was achieved in up to 83% of patients receiving an C5a antibody (IFX-1, NCT 03001622). Thus, targeting the C5a signaling may represent a promising therapeutic strategy in HS.

Most of the C5a effects result from binding to the canonical complement 5a receptor 1, C5aR1. However, there is a second C5a receptor, C5L2, whose roles are still controversial. Both pro- and anti-inflammatory properties have been proposed for C5L2. These contradictory results may be dependent on specific physiological or pathological conditions (Zhang T, 2017). ChemoCentryx has a series of potent and selective small molecule C5aR1 inhibitors (C5aRi) that are being developed for inflammatory diseases such as ANCA associated vasculitis, C3 glomerulopathy and HS. In this proposal, the investigators will use these C5aRi to examine the differential effects of targeting C5aR.

For Biopsies

An ex vivo skin culture system will be utilized to examine the effects of C5aR inhibition on neutrophil activation/inflammatory activities. Four millimeter (4mm) lesional and perilesional skin biopsies will be taken from skin excisions of 10 patients with known Hidradenitis Suppurativa. Surplus skin excision material from "deroofment" treatment of HS patients will be used. Peri-lesional and lesional biopsies will be collected for comparison. These skin biopsies will be cultured ex vivo. Analysis of the C5a-C5aR axis will be investigated by means of stimulation with recombinant C5a and/or inhibition of C5aR using a small molecule C5aR inhibitor. Briefly, each biopsy will be divided equally into 2 portions and put into culturing medium containing either C5aR inhibitor or its vehicle (DMSO). These samples will be delivered to ChemoCentryx for further analysis. Total treatment time will be 4 hours. Flow cytometry and/or immuno-histochemistry analysis will be conducted to determine the cellular changes. Cytokines in the treated biopsies or released into the culturing medium will be measured. Detailed readouts are as following:

- Immune cell population profiling (neutrophils, macrophage, T cells) by flow cytometry

- CD11b/C5aR levels on neutrophils by flow cytometry

- Complement factor (such as C1q, C4d, Bb, iC3b, C5b-9) immunohistochemistry (pending extra tissue availability after flow cytometry analysis)

- Measurement of cytokines in supernatant by multiplex ELISA: IL-1β, IL-6, TNF-α, IL-12, IL-23, IL-17A, IL-17F, IFN-γ.

These studies will extend the on-going studies with Dr. Kavita and colleagues, and will test the hypothesis that continued activation of neutrophils and other C5a-expressing leukocytes in the skin lesions of HS patients contribute to tissue damage.

For Blood Draws The investigators plan to collect plasma samples from 20 HS patients with active disease. All sample collection will follow a well-defined protocol. 20ml blood will be collected sodium citrate tubes, 5ml in EDTA tubes, and 5 ml in serum tubes. Samples will be immediately transported on ice to ChemoCentryx for processing. Blood samples will be centrifuged at 1000G for 5 minutes at 4oC, within 1 hour after collection. Plasma will be aliquoted to 0.5ml/tube and stored in -80 oC freezer immediately after centrifugation.

Before analysis, the frozen plasma samples will be thawed at room temperature and then kept on ice at all time. Concentrations of C5a, C5b-9 and C3a in these plasma samples will be measured with commercially available ELISA kits (from R&D systems, BD Biosciences, and Quadel).

All plasma samples will be used to stimulate purified from health donors. C5aRi, neutralizing antibodies against C5L2 and C5a will be employed in these neutrophil activation assays to determine the relative contribution of C5aR1 and C5L2 to the C5a-mediated activation.

Treatment conditions:

1. HS patient plasma + Vehicle of C5aRi (DMSO)

2. HS patient plasma + C5aRi

3. HS patient plasma + Isotype matched control antibody for anti-C5L2

4. HS patient plasma + anti-C5L2

5. HS patient plasma + Vehicle of C5aRi (DMSO) + Isotype matched control antibody

6. HS patient plasma + C5aRi + Isotype matched control antibody

7. HS patient plasma + Vehicle of C5aRi (DMSO) + anti-C5L2

8. HS patient plasma + C5aRi + anti-C5L2 9 HS patient plasma + Isotype matched control antibody for anti-C5a

10. HS patient plasma + anti-C5a 11. HS patient plasma 12. Normal plasma

Readouts:

CD11b surface expression on neutrophils Neutrophil degranulation / myeloperoxidase release