Abstract

The role of the TRPV1-CGRP axis in the mucosal pathophysiology of HSV-2/HIV-1 co- infection.

Background: Human Immunodeficiency Virus type 1 (HIV-1) and Herpes Simplex Virus type 2 (HSV-2) invade genital epithelia during their sexual transmission, and HSV-2 significantly increases HIV-1 acquisition during co-infection. HSV-2 lytically targets epithelial cells, establishes latency in peripheral neurons, and also targets mucosal immune cells including resident antigen-presenting Langerhans cells (LCs). LCs are also early cellular targets of HIV- 1, and rapidly capture and degrade incoming HIV-1 virions. Yet, virions escaping degradation are transferred to CD4+ T cells across LC-T cells conjugates. All mucosal epithelia are innervated by peripheral sensory neurons, including pain-transmitting nociceptors. They express the transient receptor potential vanilloid 1 (TRPV1) ion channel, whose activation by noxious stimuli (e.g. capsaicin (CP), the spicy component of the chili pepper) leads to neuropeptide release. Among these is the vasodilator and immunomodulator calcitonin gene- related peptide (CGRP). Importantly, nociceptors associate with LCs and affect their immune functions via secreted CGRP, as part of a complex mucosal neuro-immune interplay. The host laboratory discovered that CGRP also exerts antiviral effects, namely strongly inhibiting both HIV-1 transfer from LCs to CD4+ T cells and HSV-2 infection of LCs.

Objectives: i) Investigate the molecular / cellular mechanisms by which CGRP, its analogue SAX, and its inducer CP, inhibit HIV-1 and HSV-2 single and co-infections in LCs, as well as HSV-2 infection of genital epithelial cells; ii) Develop novel “mucosa-on-chip” microfluidic models, incorporating CGRP+ nociceptors with mucosal epithelial / immune cells, and explore HSV-2 mucosal spread.

Results: Our LC models consist of monocyte-derived LCs (MDLCs) and LCs isolated from inner foreskin tissues, and we showed that: 1) CGRP-mediated inhibition of HIV-1 transfer from MDLCs to CD4+ T cells involves increased expression of the transcription factor STAT4. The CGRP analogue SAX also inhibits HIV-1 transfer, both in MDLCs and inner foreskin LCs (Mariotton Front Immunol 2021). 2) Expression in LCs of langerin, i.e. the entry receptor for both HIV-1 and HSV-2, is increased by CGRP, but results in formation of atypical langerin “double trimers” that are in fact defective in viral binding (Cohen, Mariotton Mucosal Immunol 2022). 3) Like nociceptors, LCs express TRPV1, whose activation by CP induces secretion of CGRP. Such CP-induced CGRP inhibits HIV-1 transfer by inducing some, but not all, of the previously described CGRP-associated anti-HIV-1 effects. Moreover, comparing MDLCs to 2 inner foreskin tissue LCs indicates that MDLCs are closer to the newly identified langerinhighCD1ahigh epidermal LC subset, which we show to be the one affected by CGRP and CP (Mariotton PNAS 2023). 4) In a first newly developed in-vitro assay of HSV-2/HIV-1 co- infection in MDLCs, HSV-2 increases HIV-1 infection as expected. While CGRP inhibits both HSV-2 and HIV-1 single and co-infections, CP inhibits only HIV-1 infection (i.e. in line with its lack of effect on langerin expression). In parallel, CGRP and CP have no effect on HSV-2 infection of genital epithelial cell lines. 5) In a second newly developed “mucosa-on-chip” microfluidic model, mouse CGRP+ nociceptors achieve polarization, are preferentially targeted by HSV-2 and undergo HSV-2-mediated axonal degeneration. Moreover, HSV-2-infected MDLCs interact with polarized nociceptors axons, and relay HSV-2.

Conclusions: Our results provide new detailed insight on the antiviral roles of CGRP during HSV-2 and HIV-1 single and co-infections. These results could contribute to the elaboration / repositioning of CGRP/CP-based CGRP/anti-HSV-2/HIV-1 microbicides, acting on LCs and limiting simultaneously HSV-2 and HIV-1 infections, and their synergic infections.

Keywords: Langerhans cells, Langerin, HSV/HIV-1 Single and Co-infection, Calcitonin Gene- Related Peptide, Capsaicin, ‘Mucosa-on-chip’ Model 3