Germ line-encod­ed recep­tors and adap­tors sense signs of infec­tion and mount the appro­pri­ate innate immune response. While these respons­es can cur­tail infec­tion with human immuned­e­fi­cien­cy virus (HIV), aber­rant acti­va­tion of inflam­ma­tion is also linked to HIV patho­gen­e­sis. Although the mas­sive loss of CD4 T cells in the course of dis­ease pro­gres­sion has been linked to pyrop­to­sis, mol­e­c­u­lar details of HIV-induced inflam­ma­some assem­bly and oth­er fun­da­men­tal aspects of the innate immune response to HIV are not understood.
While pre­vi­ous stud­ies have most­ly relied on cytokine mea­sure­ments and indi­rect assess­ment of cell loss, we pro­pose a dif­fer­ent approach to inves­ti­gate innate immune respons­es to HIV infection:
We aim to direct­ly visu­al­ize and quan­ti­fy inflam­ma­some assem­bly and antivi­ral respons­es in human pri­ma­ry cell (mix­tures) with nov­el inflam­ma­some biosen­sors and nanobod­ies. Nanobod­ies are sin­gle domain anti­bod­ies derived from camelid heavy chain-only anti­bod­ies, which we will cus­tom gen­er­ate and equip with addi­tion­al func­tion­al­i­ties by enzy­mat­ic or genet­ic mod­i­fi­ca­tion. We will detect indi­vid­ual cells that assem­ble inflam­ma­somes or mount antivi­ral tran­scrip­tion­al respons­es by flow cytom­e­try and ImageStream analy­sis. HIV strains will be equipped with a nov­el genet­i­cal­ly-encod­ed inflam­ma­some biosen­sor that can reveal inflam­ma­some assem­bly in pro­duc­tive­ly or abor­tive­ly infect­ed pri­ma­ry cell cul­tures, includ­ing PBM­Cs and ton­sil­lar human lym­pho­cyte aggre­gate cul­tures (HLAC). This will allow us to sort respond­ing cells and ana­lyze their tran­scrip­tome. Nanobod­ies will be used to visu­al­ize the involved mol­e­c­u­lar inter­ac­tions by advanced microscopy tech­niques, iden­ti­fy nov­el inter­ac­tions, and deplete pro­teins in pri­ma­ry cells for func­tion­al analysis.
We will fur­ther dis­sect the mol­e­c­u­lar reg­u­la­tion of NLRX1, a crit­i­cal anti-inflam­ma­to­ry host fac­tor required for HIV infec­tion in myeloid cells that is upreg­u­lat­ed ear­ly in SIV-infect­ed macaques. Using cus­tom-gen­er­at­ed nanobod­ies against NLRX1, we will eval­u­ate its func­tion in HIV-infect­ed pri­ma­ry cells, ana­lyze its reg­u­la­tion by oligomer­iza­tion, post-trans­la­tion­al mod­i­fi­ca­tion, or local­iza­tion, and will iden­ti­fy nov­el inter­ac­tion part­ners by prox­im­i­ty label­ing approach­es. We pos­tu­late that NLRX1 is an intri­cate­ly reg­u­lat­ed sig­nal­ing hub, which inte­grates input to broad­ly coor­di­nate anti-inflam­ma­to­ry processes.
We will gen­er­ate, apply, and share nov­el tools to visu­al­ize inflam­ma­to­ry respons­es and their reg­u­la­tion. Our work will thus help reveal how HIV infec­tion trig­gers and avoids the innate immune sys­tem in rel­e­vant pri­ma­ry cell models.