Background/Purpose: The mechanisms utilized by B cells to execute their differentiation programs when exposed to damaging and stressful environments, which often accompany severe infections and inflammatory disorders, are poorly understood. RhoA GTPases are molecular switches, which signal by binding to key downstream effectors. In particular, RhoA signaling activates two crucial serine-threonine kinases, ROCK1 and ROCK2. In B cells, ROCK2 is known to regulate the positioning and cholesterol biosynthesis of germinal center B cells and to control plasma cell (PC) differentiation. Although ROCK1 and ROCK2 share a highly homologous kinase domain, they exhibit a lower degree of similarity in the remainder of the molecule suggesting the existence of isoform-specific functions. The precise role of ROCK1 in B cells has, however, not been studied. Here, we have investigated the contribution of ROCK1 to B cell differentiation in complex inflammatory settings by employing a malaria infection model and in vitro studies.

Methods: Mice with B cell-specific deletion of ROCK1 (CD23-Cre.Rock1^flox/flox) were subjected to Plasmodium yoelii 17XNL (P. yoelii), a non-lethal malaria infection model characterized by severe transient anemia. Rock1^flox/flox mice were employed as controls. Immune responses were monitored by FACS at various timepoints and histopathological analyses. Antibody responses and cytokine production were assessed by ELISA. The molecular mechanisms employed by ROCK1 to regulate B cell differentiation were examined by FACS-sorting B cell populations from spleens followed by RNA-sequencing analyses and in vitro experiments coupled with transcriptomic, phospho-proteomic, and biochemical strategies.

Results: In vivo and in vitro studies demonstrate that the serine-threonine kinase ROCK1 helps B cells execute their differentiation programs upon exposure to pathogen-associated stressors, like TLR9 ligands and high levels of heme. ROCK1 restrains premature plasma cell differentiation by phosphorylating the heme-regulated transcription factor Bach2. As B cells differentiate, furthermore, ROCK1 limits the proinflammatory potential of B cells, and fine-tunes mTORC1 activity and stress responses. ROCK1 orchestrates these processes by regulating the assembly of p62 complexes containing mTORC1, TBK1, RhIM-domain proteins, and molecules involved in RNA metabolism and proteostasis.

Conclusion: These studies indicate that ROCK1 is a critical “stress” sensor, which helps B cells execute their differentiation programs upon exposure to potentially damaging conditions by coordinating the activity and localization of a key core of molecules that mediate cell-fate decisions, proinflammatory functions, and RNA and protein homeostasis. These ROCK1-dependent mechanisms may be widely employed by B cells to cope with intense environmental stresses and these findings may be relevant not only for infections, but also for vaccinations, and autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rock1-orchestrates-b-cell-differentiation-under-stress/