Impact of the microbial derived short chain fatty acid propionate on host susceptibility to bacterial and fungal infections in vivo.

Short chain fatty acids (SCFAs) produced by intestinal microbes mediate anti-inflammatory effects, but whether they impact on antimicrobial host defenses remains largely unknown. This is of particular concern in light of the attractiveness of developing SCFA-mediated therapies and considering that SCFAs work as inhibitors of histone deacetylases which are known to interfere with host defenses. Here we show that propionate, one of the main SCFAs, dampens the response of innate immune cells to microbial stimulation, inhibiting cytokine and NO production by mouse or human monocytes/macrophages, splenocytes, whole blood and, less efficiently, dendritic cells. In proof of concept studies, propionate neither improved nor worsened morbidity and mortality parameters in models of endotoxemia and infections induced by gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae), gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumoniae) and Candida albicans. Moreover, propionate did not impair the efficacy of passive immunization and natural immunization. Therefore, propionate has no significant impact on host susceptibility to infections and the establishment of protective anti-bacterial responses. These data support the safety of propionate-based therapies, either via direct supplementation or via the diet/microbiota, to treat non-infectious inflammation-related disorders, without increasing the risk of infection

Sirtuin 5 Deficiency Does Not Compromise Innate Immune Responses to Bacterial Infections.

Sirtuin 5 (SIRT5) is a member of the family of NAD <sup>+</sup> -dependent lysine/histone deacetylases. SIRT5 resides mainly in the mitochondria where it catalyzes deacetylation, demalonylation, desuccinylation, and deglutarylation of lysine to regulate metabolic and oxidative stress response pathways. Pharmacologic inhibitors of SIRT5 are under development for oncologic conditions, but nothing is known about the impact of SIRT5 on antimicrobial innate immune defenses. Using SIRT5 knockout mice, we show that SIRT5 deficiency does not affect immune cell development, cytokine production and proliferation by macrophages and splenocytes exposed to microbial and immunological stimuli. Moreover, preclinical models suggest that SIRT5 deficiency does not worsen endotoxemia, Klebsiella pneumoniae and Streptococcus pneumoniae pneumonia, Escherichia coli peritonitis, listeriosis, and staphylococcal infection. Altogether, these data support the safety profile in terms of susceptibility to infections of SIRT5 inhibitors under development

High-dimensional immune phenotyping of blood cells by mass cytometry in patients infected with hepatitis C virus.

Chronic hepatitis C virus (HCV) infection affects the immune system. Whether elimination of HCV with direct-acting antivirals (DAA) restores immunity is unclear. We used mass cytometry to get a broad and in-depth assessment of blood cell populations of patients with chronic HCV prior to and after DAA therapy. Before and 12 weeks after sustained virological response to DAA therapy (SVR12), 22 cell populations were analysed by mass cytometry in blood collected from 10 healthy controls and 20 HCV patients with (10) or without human immunodeficiency virus (HIV) (10) infection. HCV infection altered the frequency of 14/22 (64%) blood cell populations. At baseline, the frequencies (median [IQR]; control, HCV, HCV/HIV) of intermediate monocytes (1.2 [0.47-1.46], 1.76 [0.83-2.66], 0.78 [0.28-1.77]), non-classical monocytes (1.11 [0.49-1.26], 0.9 [0.18-0.99], 0.54 [0.28-1.77]), conventional dendritic cells type 2 (0.55 [0.35-0.59], 0.31 [0.16-0.38], 0.19 [0.11-0.36]) and CD56 <sup>dim</sup> natural killer cells (8.08 [5.34-9.79], 4.72 [2.59-6.05], 3.61 [2.98-5.07]) were reduced by 35% to 65%, particularly in HCV/HIV co-infected patients. In contrast, activated double-negative T cells (0.07 [0.06-0.10], 0.10 [0.09-0.19], 0.19 [0.12-0.25]), activated CD4 T cells (0.28 [0.21-0.36], 0.56 [0.33-0.77], 0.40 [0.22-0.53]) and activated CD8 T cells (0.23 [0.14-0.42], 0.74 [0.30-1.65], 0.80 [0.58-1.16]) were increased 1.4 to 3.5 times. Upon stimulation with Toll-like receptor ligands, the expression of cytokines was up-regulated in 7/9 (78%) and 17/19 (89%) of the conditions in HCV and HCV/HIV patients, respectively. Most alterations persisted at SVR12. Chronic HCV and HCV/HIV infections induces profound and durable perturbations of innate and adaptive immune homeostasis

Ion Mobility–Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates

Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility–mass spectrometry (IM–MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1β. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM–MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1β, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1β monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts