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Research ArticleAgingImmunology
Open Access | 
10.1172/jci.insight.180507
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Rivera-Correa, J. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Gupta, S. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Pannellini, T. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Miele, M. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Li, Z. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Zamponi, N. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
Find articles by Giannopoulou, E. in: JCI | PubMed | Google Scholar
1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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1Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, New York, USA.
2Biological Sciences Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA.
3Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, New York, USA.
4Microchemistry & Proteomics Core at Memorial Sloan Kettering Cancer Center, New York, New York, USA.
5Hematology and Oncology Division, Weill Cornell Medicine, New York, New York, USA.
6Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
7David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA.
8Department of Medicine, Weill Cornell Medicine, New York, New York, USA.
Address correspondence to: Alessandra B. Pernis, Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York, 10021, USA. Phone: 212.606.1612; Email: pernisa@hss.edu.
Authorship note: SG and ER contributed equally to this work.
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Published February 4, 2025 - More info
Published in Volume 10, Issue 5 on March 10, 2025
AbstractThe mechanisms utilized by differentiating B cells to withstand highly damaging conditions generated during severe infections, like the massive hemolysis that accompanies malaria, are poorly understood. Here, we demonstrate that ROCK1 regulates B cell differentiation in hostile environments replete with pathogen-associated molecular patterns (PAMPs) and high levels of heme by controlling 2 key heme-regulated molecules, BACH2 and heme-regulated eIF2α kinase (HRI). ROCK1 phosphorylates BACH2 and protects it from heme-driven degradation. As B cells differentiate, furthermore, ROCK1 restrains their pro-inflammatory potential and helps them handle the heightened stress imparted by the presence of PAMPs and heme by controlling HRI, a key regulator of the integrated stress response and cytosolic proteotoxicity. ROCK1 controls the interplay of HRI with HSP90 and limits the recruitment of HRI and HSP90 to unique p62/SQSTM1 complexes that also contain critical kinases like mTOR complex 1 and TBK1, and proteins involved in RNA metabolism, oxidative damage, and proteostasis like TDP-43. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity of key controllers of B cell differentiation and stress responses. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses, and these findings may be relevant for immune-mediated and age-related neurodegenerative disorders.
Graphical Abstract
Introduction
Precise orchestration of B cell differentiation is critical for protective immunity (1). Once activated, B cells can either migrate to extrafollicular (EF) areas and differentiate into short-lived plasmablasts/plasma cells (PB/PC) or to the follicle, where they form germinal centers (GC), eventually becoming high-affinity long-lived plasma cells or memory B cells (MBCs). B cells differentiating via the EF route include B cells that express CD11c and/or T-bet, also known as ABCs, DN2, or atypical B cells (atBCs) (2, 3). Disrupting B cell differentiation is a well-known strategy employed by pathogens to evade immune defenses as observed in malaria caused by Plasmodium parasites, which is accompanied by several disturbances ranging from exuberant polyclonal plasmablast responses to enhanced accumulation of atBCs (4). These alterations result in impaired long-lasting immunity allowing for repeated infections.
Severe pathogens can leverage the complex inflammatory environment elicited during the infection to influence B cell differentiation and the establishment of protective immunity. Among the environmental stressors faced by B cells during infections are large amounts of extracellular heme, a critical damage-associated molecular pattern (DAMP) released during the hemolysis triggered by Plasmodium and several other pathogens. Interestingly, physiologic levels of heme have recently emerged as an important factor in B cell differentiation (5, 6). This is partly due to the ability of heme to bind to and promote the degradation of BACH2, a transcription factor that not only regulates the expression of the heme-metabolic enzyme HO-1 (encoded by Hmox1) but also controls B cell differentiation by repressing the expression of B lymphocyte-induced maturation protein-1 (BLIMP1; encoded by Prdm1), thus preventing premature PC differentiation (7). Besides changes in BACH2-controlled transcriptional networks, B cell terminal differentiation also requires the coordinated execution of pathways aimed at handling the increased metabolic requirements and high rate of protein synthesis needed for robust and durable antibody secretion (8). Implementation of this program occurs in distinct phases whereby an X-box-binding protein 1–independent (XBP1-independent) “anticipatory” unfolded protein response (UPR) begins in activated B cells and is followed by the classical IRE1α-XBP1–dependent UPR during the early stages of PC differentiation (9). The “anticipatory” UPR is controlled by mTOR complex 1 (mTORC1), whose activity needs to be precisely controlled during terminal B cell differentiation to ensure adequate PC formation and maximal antibody secretion. Indeed, while mTORC1 is initially critical for PC generation, persistent mTORC1 activation, as observed with activating mutations in PI3K, a crucial upstream regulator of mTORC1, leads to decreased PC survival because of impaired autophagy and increased ER stress (10, 11). The mechanisms that fine-tune mTORC1 activity in differentiating B cells are poorly understood.
Besides PI3K, mTORC1 activation also depends on the presence of amino acids, which enable the recruitment of mTORC1 to the lysosome (12). This repositioning is controlled in a complex manner and can be mediated by a docking system that relies on p62/SQSTM1 (hereafter termed p62), an adaptor that binds raptor and positions mTOR near TNF receptor associated factor 6 (TRAF6), resulting in mTOR activation via K63-linked polyubiquitination (13, 14). Recruitment and activation of mTORC1 by p62 is facilitated by the multidomain structure of p62, a feature that enables this protein to undergo phase separation, mediate the formation of membrane-less condensates, and function as a central signaling hub positioned at the intersection of pathways that regulate not only mTORC1 activation but also inflammation, autophagy, and proteostasis (15, 16). Precise coordination of these processes is critical during intense tissue damage and may be particularly important for secretory cells like PCs, which need to meet the high bioenergetic demands of antibody production even when exposed to dwindling resources and increased stress.
Severe pathogens often manipulate host defenses by targeting RhoA GTPases, whose disarming in innate cells leads to inflammasome activation because of the inhibition of their downstream effectors, the PKN1/2 kinases (17, 18). RhoA signaling also activates another key pair of serine-threonine kinases, ROCK1 and ROCK2, which are well-known controllers of cytoskeletal dynamics. Although intensely investigated in the nonhematopoietic system (19, 20), few studies, mostly focused on ROCK2, have assessed their role in B cells. In this compartment, ROCK2 is activated in response to adaptive signals such as the engagement of CD40 and regulates the proper positioning and cholesterol biosynthesis of GC B cells and PC differentiation (21, 22). These effects have been linked to the ability of ROCK2 to phosphorylate either interferon regulatory factor 8 (IRF8) or IRF4 depending on the stage of B cell differentiation (21, 22). While ROCK1 and ROCK2 share a highly homologous N-terminal kinase domain, they exhibit a lower degree of similarity in the remainder of the molecule and are encoded by different genes, suggesting that each family member can mediate specific functions. Whether ROCK1 helps coordinate B cell activation and differentiation is, however, unknown.
Here, we demonstrate that ROCK1 plays a role in controlling B cell responses in very damaging environments accompanied by high levels of pathogen-associated molecular patterns (PAMPs) and DAMPs like heme. In the absence of ROCK1, B cells exposed to these conditions exhibit altered differentiation, functional capabilities, and stress responses. Notably, absence of ROCK1 results in the aberrant assembly of p62 complexes enriched in critical kinases and molecules involved in RNA metabolism, oxidative damage, and proteostasis. ROCK1 regulates these processes by controlling 2 key heme-regulated molecules, the transcription factor BACH2 and the heme-regulated eIF2α kinase (HRI). These studies thus uncover a surprising role for ROCK1 in the regulation of pathways that enable B cells to efficiently cope with severe damaging conditions and establish durable humoral responses.
ResultsB cell ROCK1 regulates humoral responses upon immunization. Since B cells express both ROCK1 and ROCK2 (21) (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.180507DS1), we employed a genetic approach to specifically investigate the role of B cell ROCK1 in humoral responses. To this end, we generated CD23Cre.Rock1fl/fl mice (termed CD23-Rock1) and compared them with Rock1fl/fl (WT) mice. ROCK1 was efficiently deleted in B cells, and there was no compensatory increase in ROCK2 activity by in vitro kinase assays (IVKs) (Supplemental Figure 1, A–C). Except for a small decrease in marginal zone B cells, CD23-Rock1 mice displayed normal B cell populations in the bone marrow and spleen at baseline (Supplemental Figure 1, D and E). However, after intraperitoneal immunization with a T cell–dependent (TD) antigen, NP-CGG, CD23-Rock1 mice exhibited fewer GC B cells than WT mice (Supplemental Figure 1F). We also generated Rock1fl/fl mice expressing Cγ1-Cre (termed Cg1-Rock1 mice) to induce deletion during the early stages of GC B cell differentiation. Similarly to Cg1-Rock2 mice (21), TD immunization of Cg1-Rock1 mice resulted in decreased total and antigen-specific GC B cells (Figure 1, A–C). Unlike mice lacking B cell ROCK2 (21), the ratio of dark zone to light zone GC B cells was, however, not affected by the absence of ROCK1 (Supplemental Figure 1G). No significant differences in the mutation rate were observed between WT and Cγ1-Rock1 GC B cells (Supplemental Figure 1H), suggesting that ROCK1 is not required for somatic hypermutation. Cg1-Rock1 mice also exhibited a decrease in NP-specific IgG-producing antibody-secreting cells, and lower titers of NP-specific antibodies, though the ratio of high-affinity to total NP-specific antibodies was unchanged (Figure 1D and Supplemental Figure 1I). Lack of B cell ROCK1, furthermore, did not affect the T follicular helper cell/T follicular regulatory cell ratio or the frequencies of cytokine-producing T cells (Supplemental Figure 1, J and K). These data thus suggest that, similarly to ROCK2 (21), B cell ROCK1 functions in a cell-intrinsic manner to regulate optimal GC formation after immunization.
Figure 1B cell ROCK1 regulates humoral responses during TD immunization. (A–E) WT (black) and Cg1-Rock1 (orange) mice were immunized intraperitoneally (ip) with 100 mg NP-CGG for 7–28 days as indicated. Pooled quantifications of germinal center (GC) B cells (A; B220+GL7+Fas+), NP-specific B cells (B; B220+IgM–IgD–Gr1–IgG1+NP+), NP-specific GC B cells (C; B220+IgM–IgD–Gr1–IgG1+NP+CD38lo) from WT and Cg1-Rock1 mice as assessed by flow cytometry. Data pooled from 7 WT and 6 Cg1-Rock1 mice across 2 independent experiments and show mean ± SEM; P value by unpaired 2-tailed t tests. (D) ELISA data showing relative concentrations of NP<8-IgG1 and NP>25-IgG1 in the serum of the indicated mice at days 0–28 after immunization. Data pooled from 4 mice at day 14 and 8 mice from days 0, 21, and 28 per genotype across 2 independent experiments and show mean ± SEM; P value by 2-way ANOVA followed by Holm-Šídák test for multiple comparisons. (E–H) WT or CD23-Rock1 mice were immunized ip with 100 μg NP-CGG, and GC B cells (B220+GL7+CD38lo) were sorted at day 7 for bulk RNA-Seq. Data shown are from 3 independent experiments. (E) GSEA plot shows the downregulation of the REACTOME_CHOLESTEROL_BIOSYNTHESIS pathway in CD23-Rock1 GC B cells. (F) Dot plot shows the top enriched HALLMARK pathways upregulated in CD23-Rock1 GC B cells as compared with WT GC B cells at FDR < 0.1. (G) GSEA plot showing the enrichment of the HALLMARK_TNFA_signaling_via_NFKB in CD23-Rock1 GC B cells. (H) Heatmap of the z-score–scaled expression of genes enriching the HALLMARK Inflammatory response pathway in CD23-Rock1 GC B cells. *P value < 0.05, **P value < 0.01, ***P value < 0.001, and ****P value < 0.0001.
To gain insights into the mechanisms employed by ROCK1 to control GC formation, we next performed bulk RNA-Seq on sorted GC B cells from immunized WT and CD23-Rock1 mice (Supplemental Figure 1L). Key GC markers were comparable in WT and CD23-Rock1 GC B cells (Supplemental Figure 1M). Similar to CD23-Rock2 GC B cells (21), gene set enrichment analysis (GSEA) revealed that the only downregulated pathway (FDR < 0.1) in CD23-Rock1 GC B cells was cholesterol biosynthesis (Figure 1E), indicating that both ROCKs participate in the control of this critical metabolic pathway in GC B cells. In contrast to the selective downregulation of only 1 major pathway, lack of ROCK1 led to the upregulation of several pathways in GC B cells (Figure 1F). Some of these pathways, such as HALLMARK-Epithelial Mesenchymal Transition, were related to the known cytoskeletal role of the ROCKs. Interestingly, the most upregulated pathways in CD23-Rock1 GC B cells included several pro-inflammatory pathways such as HALLMARK-Inflammatory response and HALLMARK-TNFA_signaling_via_NFKB and several targets related to inflammation (e.g., Ccl22) (Figure 1, F–H, and Supplemental Figure 1L). These findings, thus, surprisingly suggest that ROCK1 may limit the pro-inflammatory profile of GC B cells.
B cell ROCK1 regulates humoral responses and pathology during experimental malaria. The enhanced pro-inflammatory profile of ROCK1-deficient B cells upon a mild challenge like immunization led us to assess how B cell ROCK1-deficient mice would respond to a more complex and hostile milieu. We opted to employ Plasmodium yoelii 17XNL (P. yoelii), a nonlethal self-healing malaria model, which, in C57BL/6 mice, leads to RBC destruction and hemolysis, severe anemia, and parasitemia mimicking features observed in malaria-naive individuals infected with human Plasmodium species (23–25). An assessment of ROCK activity revealed that WT B cells increased ROCK1, but not ROCK2, activation as a physiologic response to this parasite at acute day 9 postinfection (pi) (Figure 2A and Supplemental Figure 2A). We next infected WT and CD23-Rock1 mice with P. yoelii and analyzed them at acute day 9 pi and at late day 21 pi when mice are normally in a convalescent phase. Although parasitemia levels were similar at day 9, lack of B cell ROCK1 impaired resolution of the infection at day 21 (Figure 2B). Total splenic B cells decreased to a greater extent in CD23-Rock1 than in WT mice at day 9 pi and did not recover as readily at day 21 pi, and GC B cells were significantly reduced at both time points (Figure 2, C–E). Expansion of atBCs at day 21 pi was unaffected by the absence of B cell ROCK1, resulting in a relative increase in atBCs over GC B cells at day 21 pi (Figure 2, F and G). Only minor decreases in total CD4+ and TFH cells were observed (Supplemental Figure 2, B and C). Despite a comparable expansion of PB/PCs at day 9 pi in WT and CD23-Rock1 mice, CD23-Rock1 mice exhibited marked decreases in total IgG1 and IgG2c at both day 9 and day 21 and in total IgM at day 21 (Figure 2, H and I). Absence of B cell ROCK1 also resulted in lower titers of anti-malaria IgG1 antibodies but not of anti-malaria IgG2c antibodies, an isotype classically produced by atBCs (Figure 2J). Thus, in this malaria model, B cell ROCK1 is important for resolution of the infection, and its absence affects B cell differentiation and the robust polyclonal antibody responses known to accompany this infection (24, 25).
Figure 2ROCK1 controls humoral responses to Plasmodium infection. WT (black) or CD23-Rock1 (orange) mice were infected with 106Plasmodium yoelii 17XNL-infected erythrocytes. Uninfected (u) or infected mice at the indicated times pi were analyzed. (A) ROCK1 and ROCK2 activity in purified CD23+ splenic B cells at day 9 pi. Immunoblotting shows p-MYPT1 in IVKs and total ROCK1 and ROCK2 in inputs. Data representative of 3 independent experiments. n.s., nonspecific band; p-, phosphorylated. (B) Parasitemia. Data from at least 5 mice per day and per genotype across 3 independent experiments and show mean ± SEM; P value by nonparametric Mann-Whitney test between the 2 genotypes for each day of infection. (C) Total splenocyte numbers. (D–H) B cell populations by FACS. Frequencies (symbols) and total cell numbers (bars) of splenic B cells (CD19+) (D), GC B cells (B220+GL7+Fas+) (E), atBCs (B220+CD11c+T-bet+) (F), PB/PC (B220int CD138+) (H). (G) Ratio of atBC to GC B cells. Data from at least 5 mice per day and per genotype across 3 independent experiments and show mean ± SEM; P value by unpaired 2-tailed t tests. (I–K) Total IgM, IgG1, and IgG2c levels (I); anti-malaria IgM, IgG1, and IgG2c relative levels (J); and CCL22 and CCL5 levels (K) in the sera by ELISA. Data from at least 5 mice per day and per genotype across 3 independent experiments and show mean ± SEM; P value by 2-way ANOVA followed by Holm-Šídák test for multiple comparisons. (L–O) RNA-Seq of splenic B cell populations sorted from WT or CD23-Rock1 mice at day 9 pi. Data are from 3 independent experiments. (L and M) Top enriched HALLMARK pathways upregulated in CD23-Rock1 compared with WT for activated B cells (L) and PB/PCs (M) at FDR < 0.1. (N and O) GSEA enrichment plots representing selected downregulated pathways in CD23-Rock1 activated B cells (N) and PB/PCs (O). *P value < 0.05, **P value < 0.01, ***P value < 0.001, and ****P value < 0.0001.
The increased pro-inflammatory profile of CD23-Rock1 GC B cells also prompted us to specifically assess the production of CCL22 and CCL5 in response to P. yoelii infection. CD23-Rock1 exhibited significantly higher serum levels of these chemokines than WT mice at both day 9 and day 21 pi (Figure 2K). CD23-Rock1 mice furthermore developed more s
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