Classical complement and inflammasome activation converge in CD14highCD16- monocytes in HIV associated TB-immune reconstitution inflammatory syndrome

Inflammasome-derived cytokines, IL-1 beta and IL-18, and complement cascade have been independently implicated in the pathogenesis of tuberculosis (TB)-immune reconstitution inflammatory syndrome (TB-IRIS), a complication affecting HIV+ individuals starting antiretroviral therapy (ART). Although sublytic deposition of the membrane attack complex (MAC) has been shown to promote NLRP3 inflammasome activation, it is unknown whether these pathways may cooperatively contribute to TB-IRIS. To evaluate the activation of inflammasome, peripheral blood mononuclear cells (PBMCs) from HIV-TB co-infected patients prior to ART and at the IRIS or equivalent timepoint were incubated with a probe used to assess active caspase-1/4/5 followed by screening of ASC (apoptosis-associated speck-like protein containing a CARD domain) specks as a readout of inflammasome activation by imaging flow cytometry. We found higher numbers of monocytes showing spontaneous caspase-1/4/5(+)ASC-speck formation in TB-IRIS compared to TB non-IRIS patients. Moreover, numbers of caspase-1/4/5(+)ASC-speck(+) monocytes positively correlated with IL-1 beta/IL-18 plasma levels. Besides increased systemic levels of C1q and C5a, TB-IRIS patients also showed elevated C1q and C3 deposition on monocyte cell surface, suggesting aberrant classical complement activation. A clustering tSNE analysis revealed TB-IRIS patients are enriched in a CD14(high)CD16(-) monocyte population that undergoes MAC deposition and caspase-1/4/5 activation compared to TB non-IRIS patients, suggesting complement-associated inflammasome activation during IRIS events. Accordingly, PBMCs from patients were more sensitive to ex-vivo complement-mediated IL-1 beta secretion than healthy control cells in a NLRP3-dependent manner. Therefore, our data suggest complement-associated inflammasome activation may fuel the dysregulated TB-IRIS systemic inflammatory cascade and targeting this pathway may represent a novel therapeutic approach for IRIS or related inflammatory syndromes. Author summary Tuberculosis (TB) associated-immune reconstitution inflammatory syndrome (TB-IRIS) is a clinical complication affecting HIV+ individuals previously co-infected with Mycobacterium tuberculosis (Mtb), upon antiretroviral therapy (ART) initiation. TB-IRIS is characterized by an exacerbated inflammatory response and can be associated with high morbidity and mortality rates in resource-limited countries with high TB prevalence. So far, there is no targeted TB-IRIS therapy, and corticosteroids are frequently used to prevent or alleviate IRIS related-symptoms. Here we found inflammasome activation (i.e. caspase1/4/5(+)ASC speck complex formation) on circulating classical CD14(high)CD16(-) monocytes may contribute to TB-IRIS immunopathology, since it correlates with pro-inflammatory cytokine plasma levels and its decay is associated with dampening in IRIS-related symptoms promoted by anti-inflammatory therapy. We also found TB-IRIS monocytes display higher surface complement deposition, being more sensitive to external complement-mediated NLRP3 inflammasome activation than healthy control cells. In fact, complement MAC molecule C9 and caspase-1/4/5 activation were associated on classical monocytes in TB-IRIS patients, suggesting complement-mediated inflammasome activation may lead to a positive feedback loop in the inflammatory responses observed in TB-IRIS. Therefore, our findings support that complement-NLRP3/ASC/caspase1/4/5 axis may be considered as a potential target for host-directed therapy of TB-IRIS.

Automated summary

In TB-IRIS patients, inflammasome activation on monocytes is associated with pro-inflammatory cytokine levels, while complement deposition may lead to a positive feedback loop in the inflammatory responses observed.